2009 NJC H2 Bio Prelims P3 EQ4 answers and examiner%27s comments

2009年普通高等学校招生全国统一考试全国2卷理科综合本试卷分第Ⅰ卷（选择题）和第Ⅱ卷(非选择题)两部分。

第Ⅰ卷1至5页，第Ⅱ卷6至14页。

考试结束后,将本试卷和答题卡一并交回.第Ⅰ卷选择题共126分一、选择题（本题共13小题，在每小题给出的四个选项中，只有一项是符合题目要求的.）本卷共21小题，每小题6分，共126分。

一、选择题(本题共13小题。

在每小题给出的四个选项中，只有一项是符合题目要求的。

）1.下列关于细胞呼吸的叙述，错误的是A。

细胞呼吸必须在酶的催化下进行B.人体硬骨组织细胞也进行呼吸C。

酵母菌可以进行有氧呼吸和无氧呼吸D。

叶肉细胞在光照下进行光合作用，不进行呼吸作用2。

人体甲状旁腺分泌甲状旁腺素，当人体血钙浓度下降时,甲状旁腺素分泌增加，作用于骨和肾脏使血钙浓度上升。

甲状腺C细胞分泌降钙素，当血钙浓度上升时，降钙素分泌增加，作用于骨等使血钙浓度下降。

下列关于血钙的叙述，错误的是A。

血钙浓度降低可引起鸡肉抽搐B.甲状旁腺素和降钙素对血钙的调节表现为协同作用C。

人体血钙浓度在体液调节下处于动态平衡D.食物中的钙可以通过消化道吸收进入血液3.下列有关哺乳动物个体发育的叙述,错误的是A。

胚胎发育过程中也会出现细胞衰老B.幼鹿经过变态发育过程长出发达的鹿角C.胚后发育过程中伴有细胞分化D.来自原肠胚同一胚层的细胞经分化发育成不同的组织4.为防止甲型H1N1病毒在人群中的传播，有人建议接种人流感疫苗,接种人流感疫苗能够预防甲型H1N1流感的条件之一是：甲型H1N1病毒和人流感病毒具有相同的A。

侵染部位B。

致病机理C。

抗原决定簇D。

传播途径5。

下列属于种间竞争实例的是A.蚂蚁取食蚜虫分泌的蜜露B。

以叶为食的菜粉蝶幼虫与蜜蜂在同一株油菜上采食C.细菌与其体内的噬菌体均利用培养基中的氨基酸D.某培养瓶中生活的两种绿藻，一种数量增加，另一种数量减少6.物质的量之比为2：5的锌与稀硝酸反应，若硝酸被还原的产物为N2O,反应结束后锌没有剩余,则该反应中被还原的硝酸与未被还原的硝酸的物质的量之比是A. 1:4 B。

Fisher KernelMartin Sewell2007Jaakkola and Haussler(1999a)introduced the Fisher kernel(named in hon-our of Sir Ronald Fisher),thus creating a generic mechanism for incorporat-ing generative probability models into discriminative classifiers such as SVMs. Jaakkola and Haussler(1999b)introduced a generic class of probabilistic re-gression models and a parameter estimation technique that can make use of arbitrary kernel functions.Jaakkola,Diekhans and Haussler(1999)applied the Fisher kernel method to detecting remote protein homologies which performed well in classifying protein domains by SCOP superfamily.Jaakkola,Diekhans and Haussler(2000)found that using the Fisher kernel significantly improved on previous methods for the classification of protein domains based on remote homologies.Moreno and Rifkin(2000)used the Fisher kernel method for large scale Web audio classification.Mika,Smola and Sch¨o lkopf(2001)presented a fast training algorithm for the kernel Fisher discriminant classifier.It improved upon the state of the art by more than an order of magnitude,thus rendering the kernel Fisher algorithm a viable option also for large datasets.Vinokourov and Girolami(2001)successfully employed the Fisher kernel for document classifi-cation.Saunders,Shawe-Taylor and Vinokourov(2003)showed how the string kernel can be thought of as a k-stage Markov process,and as a result interpreted as a Fisher kernel.Tsuda,et al.(2004)analyzed the statistical properties of the Fisher kernel.Nicotra,Micheli and Starita(2004)extended the Fisher kernel to deal with tree structured data.Kersting and G¨a rtner(2004)extend the Fisher kernel to logical sequences(sequences over an alphabet of logical atoms).Their experiments showed a considerable improvement over results achieved without Fisher kernels for logical sequences.Holub,Welling and Perona(2005)suc-cessfully combined generative models with Fisher kernels to realize performance gains on standard object recognition data-sets.The log-likelihood of a data item x with respect to the model m(θ0)for a given setting of the parametersθ0is defined to belog Lθ0(x).Consider the vector gradient of the log-likelihoodg(θ,x)=∂log Lθ∂θiNi=1.1The Fisher score of a data item x with respect to the model m(θ0)for a given setting of the parametersθ0isg(θ0,x).The Fisher information matrix with respect to the model m(θ0)for a given setting of the parametersθ0is given byI M=E[g(θ0,x)g(θ0,x) ],where the expectation is over the generation of the data point x according to the data generating distribution.The Fisher score gives us an embedding into the feature space R N and hence immediately suggests a possible kernel.The matrix I M can be used to define a non-standard inner product in that feature space.The invariant Fisher kernel with respect to the model m(θ0)for a given setting of the parametersθ0is defined asg(θ0,z).κ(x,z)=g(θ0,x) I−1MThe practical Fisher kernel is defined asκ(x,z)=g(θ0) g(θ0,z).The Fisher kernel gives a“natural”similarity measure that takes into ac-count an underlying probability distribution.It seems natural to compare two data points through the directions in which they‘stretch’the parameters of the model,that is by viewing the score function at the two points as a function of the parameters and comparing the two gradients.If the gradient vectors are similar it means that the two data items would adapt the model in the same way,that is from the point of view of the given parametric model at the current parameter setting they are similar in the sense that they would require similar adaptations to the parameters.ReferencesHOLUB,Alex D.,Max WELLING,and Pietro PERONA,bining generative models andfisher kernels for object recognition.In:Proceedings of the Tenth IEEE International Conference on Computer Vision(ICCV’05), Volume1.Washington,DC,USA:IEEE,pp.136–143.JAAKKOLA,Tommi,Mark DIEKHANS,and David HAUSSLER,ing the Fisher kernel method to detect remote protein homologies.In:Thomas LENGAUER,et al.,eds.Proceedings of the Seventh International Conference on Intelligent Systems for Molecular Biology.Menlo Park,CA:AAAI Press, pp.149–158.JAAKKOLA,Tommi,Mark DIEKHANS,and David HAUSSLER,2000.A discriminative framework for detecting remote protein homologies.Journal of Computational Biology,7(1–2),95–114.2JAAKKOLA,Tommi S.,and David HAUSSLER,1999a.Exploiting genera-tive models in discriminative classifiers.In:Michael S.KEARNS,Sara A. SOLLA,and David A.COHN,eds.Advances in Neural Information Pro-cessing Systems11,Bradford Books.Cambridge,MA,USA:The MIT Press, pp.487–493.JAAKKOLA,Tommi S.,and David HAUSSLER,1999b.Probabilistic kernel regression models.In:David HECKERMAN and Joe WHITTAKER,eds. Proceedings of the1999Conference on AI and Statistics.San Mateo,CA: Morgan Kaufmann.KERSTING,Kristian,and Thomas G¨ARTNER,2004.Fisher kernels for logical sequences.In:Jean-Fran¸c ois BOULICAUT,et al.,eds.Machine Learning: ECML2004:15th European Conference on Machine Learning,Pisa,Italy, September2004,Proceedings.Berlin/Heidelberg:Springer,pp.205–216. MIKA,Sebastian,Alexander SMOLA,and Bernhard SCH¨OLKOPF,2001.An improved training algorithm for kernelfisher discriminants.In:Tommi JAAKKOLA and Thomas RICHARDSON,eds.Artificial Intelligence and Statistics2001:Proceedings of the Eighth International Workshop:January 4-7,2001Key West,Florida.San Francisco,CA:Morgan Kaufmann,pp.98–104.MORENO,Pedro J.,and Ryan RIFKIN,ing the Fisher kernel method for Web audio classification.In:2000IEEE International Conference on Acoustics,Speech,and Signal Processing:Proceedings,Volume IV.IEEE, pp.2417–2420.NICOTRA,Luca,Alessio MICHELI,and Antonina STARITA,2004.Fisher kernel for tree structured data.In:Proceedings:2004IEEE International Joint Conference on Neural Networks,Volume3.IEEE,pp.1917–1922. SAUNDERS,Craig,John SHAWE-TAYLOR,and Alexei VINOKOUROV, 2003.String kernels,fisher kernels andfinite state automata.In:Suzanna BECKER,Sebastian THRUN,and Klaus OBERMAYER,eds.Advances in Neural Information Processing Systems15,Bradford Books.Cambridge,MA: The MIT Press,pp.633–640.TSUDA,Koji,et al.,2004.Asymptotic properties of the Fisher kernel.Neural Computation,16(1),115–137.VINOKOUROV,Alexei,and Mark GIROLAMI,2001.Document classifica-tion employing the Fisher kernel derived from probabilistic hierarchic corpus representations.In:Proceedings of ECIR-01,23rd European Colloquium on Information Retrieval Research.British Computer Society Information Re-trieval Specialist Group.Berlin:Springer Verlag,pp.24–40.3。

P R O T O C O L1Protein extraction from Tissues and Cultured Cells using Bioruptor ® Standard & PlusIntroductionProtein extraction from tissues and cultured cells is the first step for many biochemical and analytical techniques (PAGE, Western blotting, mass spectrometry, etc.) or protein purification. Efficient disruption and homogenization of animal tissues and cultured cells are required to ensure high yields of proteins. Diagenode’s Bioruptor ® uses state-of-the-art ultrasound technology to efficiently disrupt and homogenize tissues and cultured cells in just one step. Bioruptor ® offers unique benefits for tissue disruption and homogenization:• Fast and simple• No contamination between samples • Efficient• Gentle processing • Reproducible• Temperature controlled • Multiplexing capabilityGeneral remarks before starting• Conditions for protein extraction (e.g. use of fresh or frozen tissue, composition of extractionbuffer etc.) must be adjusted according to the nature of the proteins of interest and the assays to be run. SDS might be added to the extraction buffer to maximize the yield of soluble proteins. SDS extracts can be used for SDS electrophoresis and Western blotting. It is recommended to reduce the SDS concentration for 2D electrophoresis, enzyme-linked immunosorbent assay and mass spectrometry.• For functional studies (e.g. the study of protein–protein interactions), avoid using ionicdetergents and high concentrations of salt.Extraction buffer: use RIPA buffer as a starting point for optimization:50 mM Tris-HCl (pH 7.4) 150 mM NaCl 1% NP-400.25% Na-deoxycholate Protease Inhibitor Mix SDS 0.1 - 2% (optional)It is always recommended to optimize the buffer composition depending on a specific research project• Always use Protease Inhibitor Mix during extraction procedure to block the possible proteindegradation.P R O T O C O L@@@@2• Use Diagenode’s TPX tubes for sonication. Depending on the desired final volume, 1.5 ml TPXmicrotubes (Cat. No.: C30010010-50 or C30010010-1000) or 15 ml TPX tubes (Diagenode, Cat. No.: C30010009) might be used. Always respect the recommended sonication volumes: 100 - 300 µl for 1.5 ml TPX tubes and 1 - 2 ml for 15 ml TPX tubes (strictly follow the Bioruptor ® instructions as shown in the corresponding manual before starting any sonication experiments).• Keep extracted proteins at -80°C.Required materials and reagents• Bioruptor ® Standard (Cat. No. B01010001) or Plus (Cat. No. B01020001)• Water Cooler (Cat. No. B02010003; 115V or B02010002; 230 V)• Single Cycle Valve (Cat. No. B02020004) (required for Bioruptor ® Plus)• Tube holder for 1.5 ml tubes (Cat. No. B01200011)• 15 ml sonication accessories for Bioruptor ® Standard & Plus (Cat. No. B01200015)•Choose between option :A 1: 1.5 ml (Cat. No. C30010010-50 or C30010010-1000) or 15 ml TPX tubes (Cat. No. C30010009) for sonicationA 2:P rotein Extraction Beads (Cat No. C20000021) for tissue disruption (not required for cell lysis )B: Protein Extraction kit (Cat. No. C20000020); pre-filled 15 ml TPX tubes • Protease Inhibitor Mix (Cat. No. C12010011 or C12010012)• Buffer for protein extraction from tissue or cell lysis (not supplied)•Reagents for protein quantification (optional)ProtocolI. Protein extraction from Tissues»This protocol has been validated for up to 50 mg of tissue. Do not use more tissue per sample. For larger quantity cut the tissue and proceed to the disruption in separate tubes. When proceeding 20 - 50 mg of tissue 15 ml TPX tubes are recommended with a final volume of 1 - 2 ml. Less tissue could be sonicated in 1.5 ml TPX tubes with a final volume of 100 - 300 µl. »Minimize the time of tissue collection to prevent protein degradation.»Dissected tissues can be snap-frozen in liquid nitrogen and stored at -80°C until protein extraction1. Pre-cool Bioruptor ® to 4°C using the Bioruptor ® Water Cooler (Diagenode, Cat. No. BioAcc-Cool).2. Fill the TPX tubes with Protein Extraction Beads.»The recommended quantity of the beads is 200 - 250 mg for 15 ml TPX tubes, 40 - 50 mg for 1.5 ml TPX tubes. Note: I f using pre-filled tubes (Cat. No. C20000020 Protein Extraction kit) pleaseskip this step!3. Add Protease Inhibitor Mix (200x) to the cold protein extraction buffer: 5 µl per 1 ml of extractionbuffer. Scale accordingly.P R O T O C O L34. Add the required volume of a cold extraction buffer to the TPX tubes filled with Protein ExtractionBeads. 5. Add tissue pieces to the TPX tubes. Make sure that the final volume is in the recommendedrange: 100 - 300 µl for 1.5 ml TPX microtubes and 1 - 2 ml for 15 ml TPX tubes. 6. Vortex tubes briefly and proceed to sonication by using the Bioruptor ® with the following settings:Power: H position (High)Sonication cycle: 30 sec ON/30 sec OFF Total sonication time: 5 - 15 cycles Temperature: 4°C»To guarantee homogeneity of sonication, the tube holder should be always completely filled with tubes.7. Stop the Bioruptor ® after each 5 cycles, vortex samples and check the sample visually fordisruption.»Please note that the optimization might be required depending on the sample format (fresh or frozen tissue), tissue type and tissue amount. The shortest sonication time should be chosen to prevent protein damage. Incomplete disruption may occur with fibrous tissues (i.e. muscles). 8. Transfer the supernatant to a new tube and centrifuge samples at 14,000 rpm for 15 min at 4°Cto remove any remaining insoluble material.»The Protein Extraction Beads might be washed once with extraction buffer for maximum recovery of total protein but this will lead to the sample dilution. 9. Transfer the supernatant containing soluble proteins to a new tube.10. Take an aliquot for the quantification and the further analysis if needed. Store proteins extractsin small aliquots at -80°C.»Different protein concentration assays exist including: absorbance at 280 nm, Lowry Assay, Bradford Assay, Bicinchoninic Assay (BCA) etc.. Many commercial kits for protein quantification are also available. Please note that measuring the protein concentration in an SDS extract requires that the assay is compatible with the detergent and reducing agent in the solution. II. Protein extraction from Cultured Cells»This protocol has been validated using RIPA buffer but it may be necessary to optimize the buffer composition depending on a specific research project.»We recommend using 100 µl of an appropriate lysis buffer per 1x10^6 cells.»For Western blotting, cells might be lysed directly in 1x Laemmli buffer. After sonication, centrifuge extract at 14,000 rpm for 15 min. Transfer the supernatant to a new tube and boil for 3 min. The supernatant can be used in Western blot. Note that protein quantification by common methods is not compatible with Laemmli buffer.1. Pre-cool Bioruptor ® to 4°C using the Water Cooler.2. A dd Protease Inhibitor Mix (200x) to the ice-cold cell lysis buffer: 5 µl per 1 ml of extraction buffer .Scale accordingly.P R O T O C O L@ / info @ // North America - Diagenode Inc. / orders.na @ / info.na @43. For monolayer cells:R inse the monolayer cells 3 times with cold PBS. For the final rinse, use a cell scraper and transfer the cell suspension to a TPX tube. Centrifuge cells at 1,500 rpm for 10 min at 4°C and aspirate as much supernatant as possible. Proceed to step 4.For suspension cells:C entrifuge suspension at 1,500 rpm for 10 min at 4°C and aspirate the supernatant. Resuspend the pellet in cold PBS, transfer to a TPX tube and centrifuge at 1,500 rpm for 10 min at 4°C. Aspirate the supernatant. Repeat 2 more times. Proceed to the step 4.4. Add ice-cold cell lysis buffer and resuspend the pellet. Incubate on ice for 10 min.»The viscosity may appear at this step5. Vortex tubes briefly and proceed to sonication by using the Bioruptor ® with the following settings:Power: H position (High)Sonication cycle: 30 sec ON/30 sec OFF Total sonication time: 5-10 cycles Temperature: 4°C»To guarantee homogeneity of sonication, the tube holder should be always completely filled with tubes.6. Stop the Bioruptor ® after 5 cycles, briefly vortex samples and visually check the samples:Samples should be in solution (viscosity should be reduced)»Please note that the optimization might be required depending on sample format (cell density, cell type etc.). The shortest sonication time should be chosen to prevent protein damage. 7. Transfer the supernatant to a new tube and centrifuge samples at 14,000 rpm for 15 min at 4°Cto remove any remaining insoluble material.8. Take an aliquot for the quantification and the further analysis if needed. Store protein extractsat -80°C.»Different protein concentration assays exist including: absorbance at 280 nm, Lowry Assay, Bradford Assay, Bicinchoninic Assay (BCA) etc. Many commercial kits for protein quantification are also available. Please note that measuring the protein concentration in an SDS extract requires that the assay is compatible with the detergent and reducing agent in the solution.Figure 1. Protein Extraction Beads are required for efficient tissue disruption using the Bioruptor® PlusComplete disruption is observed in the sample containing Diagenode’s Protein Extraction Beads (left) after 5 cycles while non-disrupted tissue is still present in the sample without the Protein Extraction Beads (right).P R O T O C O L / info @ // North America - Diagenode Inc. / orders.na @ / info.na @5Figure 2. Total proteins effectively extracted from tissues using Bioruptor ® PlusVarious mouse tissues were disrupted in RIPA buffer supplemented with or without 2% SDS. Total proteins were separated by SDS-PAGE and stained with Coomassie Blue dye.Figure 3. Western blot analysis of GAPDH and HSP90 proteins in tissue and cultured cell extracts.Expected bands of 37 kD and 90 kD are observed for GAPDH (left panel) and HSP90 (right panel), respectively, in liver, brain and skeletal muscle. Note that HSP90 is expressed in muscle in an extremely low level (H. Quraishi and I. R. Brown, J Neurosci Res. 1996 Feb 1;43(3):335-45). Whole cell extract from HeLa cells is loaded as positive control. HeLa cells were lysed using the Bioruptor ®.P R O -P R O TE I N -E X T R A C T _06_08_13LiverBrainMuscle+ SDS+ SDS + SDS- SDS- SDS- SDS。

5973扩散泵经历了返油严重污染，迫不得也清洗离子源，四级杆和所有真空环境下的零件。

如下图：先申明，这个帖子不是写出来让大家都来拆仪器的哦，要有十足的把握才能做（建议至少对各部件原理及构造闭着眼睛都能把握，我是这样认为的），要不然损失可能会很大。

下图是四级杆，离子源，扩散泵的全拆合影图，全部都用棉签擦过后拍的照。

下面垫了不起毛的布，防止磨损和滑动造成的损伤。

拆机注意事项，一关二放！关就是关闭所有的电源并断开，“放”指的是放静电，拆机的时候最好带上防静电手环，不要对任何部件造成人为的损伤。

总的来说做到胆大心细，胸有成竹就可以了。

也是合影，多了工具包和氧化铝，右上角是扩散泵的散热风扇，左下角的杯子里是氧化铝粉末，用来打磨离子源的。

离子源组件的全家福（EI源）它工作原理是样品从接口螺母进入离子源，灯丝发射的电子在永磁体磁场的作用下进入离子化室，高能的电子与样品的分子作用，使得发生电离，推斥极上的正电压把正离子推向透镜组，离子被聚焦，加速聚集成离子束进入质量分析器。

磁场组件的全家福，不包括中间的黄色小螺丝刀。

磁铁是放在离子化室的两边的，用来校准电子运动的方向。

四级杆组件全家福四级杆和加热组件检测器、固定四级杆的陶瓷环离子源套筒离子源套筒侧面四级杆套筒，立四级杆套筒，测安捷伦的双曲面镀金石英四级杆，横四级杆，立四级杆接线块,向四级杆施加直流和射频电压，产生一个动态的电场。

我们把这个动态的电场叫做四级场，能满足稳定震荡的离子才能通过四级场，仪器通过精确控制电压变化，使得质荷比的离子通过动态电场到达检测器，在电压变化每一极短时间内，只有一种质荷比的离子可以通过去，达到质量过滤器的作用，永磁体永磁体基座铝制传热块四级杆加热组件，四级杆加热组件的传感器非惰性的离子源体真空腔体前端，左边放空阀，右边调谐液网印技术∙个人资料加为好友∙给他留言帖子合集1# 只看作者回复于：2009/11/3 11:16:20回复本贴回复主题编辑举报管理下面三张图是扩散泵，也叫做蒸汽喷射泵，它的工作原理是，先通过前级泵抽得粗真空，使得扩散泵的入口处被抽成真空系统，扩散泵的底部是一个加热的炉盘，它把泵油加热到沸腾，泵油蒸汽附着在泵体中心的由同心圆筒状组成的泵心上升，但是其向下斜的斜边设计就使得蒸汽向下喷射，动能就会传递给气体分子，使得气体分子向底部走，并被前级泵从排气口抽走。

免疫学技术复习思考题及答案复习思考题：1.免疫细胞分离的基木原理（密度、黏附吞噬、表面标志、生长因子）应用于哪些细胞？2.细胞因子检测的三大基本方法（原理、优缺点）3.FACS法测CD分子的基本原理4.MACS法分离细胞的原理5.ELISA和ELISPOT实验原理6.T细胞增殖试验的基木原理、基本方法7.细胞凋亡的测定原理和方法8.概括免疫学技术主要内容9.单克隆抗体制备的原理和基木过程10.凝集、沉淀反应的主要方法（名称和基本原理）11.各种免疫标记技术的标记物及示踪原理12.免疫荧光技术的各种实验策略13.ELISA的基木原理利各种实验策略14.Western blot的基本原理1.E LISA技术原理和实验策略ELISA全名为酶联免疫吸附试验。

原理是将抗原或抗体包被在固相载表面，并保留其免疫活性，然后再与前标抗体（或酶标抗原）联结，并保留酮活性.洗去游高的酶标抗体（或酶标抗原），然后加入酶反应的底物，底物被酶催化变成石色产物。

反映颜色深浅与相应的抗原或抗体的量相关，据此进行定性和定量分析°主要的实验策略有：双抗体夹心法：此法适用于检验各种蛋白质等大分子抗原。

将待分析物的特异性抗体与固相载体连接形成固相抗体，经封闭和洗涤后加受检标本（抗原）形成固相抗体一抗原复合物，洗涤后再加前标抗体生成抗体-待测抗原-酶标记抗体的复合物，洗涤利底物显色。

根据颜色反应的程度进行该抗原的定性或定量测定双位点一步法：在双抗夹心基础上，改为应用针对抗原分子上两个不同决定簇的两种单克隆抗体分别作为固相抗体和酶标抗体。

其优点是测定时标木可与酶标抗体同时加入进行结合反应，两种抗体互不干扰。

省时简便。

但是当待测抗原浓度过高时，可出现假阴性结果。

间接法：检测抗体最常用的方法，其原理为利用酶标记的抗抗体以检测已与固相结合的受检抗体，故称为间接法。

用已知抗原包被固相载体，加待检标木与固相抗原结合，洗涤后加酶标抗抗体与固相载体上抗原抗体复合物结合，经洗涤，底物显色，根据颜色反应的程度进行该抗体的定性或定量测定竞争法：此法可用于抗原和半抗原的定量测定，也可用于测定抗体。

Procedure & Checklist – Preparing HiFi SMRTbell®Libraries using the SMRTbell Express Template Prep Kit 2.0This procedure describes the construction of HiFi SMRTbell libraries for de novo assembly and variant detection applications using the SMRTbell Express Template Prep Kit 2.0 and recommended HiFi sequencing conditions using PacBio’s new Sequel® II Binding Kit 2.2. A minimum input amount of 5 µg of high-molecular weight genomic DNA is recommended for generating HiFi library yields sufficient for running multiple SMRT®Cells on the Sequel II or Sequel IIe System (Sequel II Systems). Note that final HiFi library construction yields will be dependent on the specific size-selection method employed.We recommend fragmenting the gDNA so that the target size distribution mode is between 15 kb - 18 kb. To reduce the presence of fragments >30 kb, PacBio recommends a 2-cycle shearing method on the Megaruptor 3 system. Generally, a narrower fragment size distribution results in more uniform and higher-quality HiFi data. Details regarding DNA shearing conditions (e.g., buffers and DNA sample concentration) are described in the “DNA Requirements for Shearing” section.RequiredEquipment Vendor Throughput Run TimeFemto Pulse AgilentTechnologies Process up to 11 samples per runBatch process up to 88 samples 85 minsMegaruptor 3 Diagenode Shear up to 8 samples at a time40 mins(for 1 cycle of shearing)PippinHT Sage Science Maximum of 20 samples per instrument run 2 hrsBluePippin Sage Science Maximum of 4 samples per instrument run 4.5 hrsSageELF Sage Science Maximum of 2 samples per instrument run 4.5 hrs Table 1: Recommended equipment for HiFi SMRTbell library construction for de novo assembly and variant detection applications.Required MaterialsDNA SizingFemto Pulse Agilent Technologies, Inc. P-0003-0817DNA QuantitationQubit™ Fluorometer ThermoFisher Scientific Q33238Qubit 1X dsDNA HS Assay Kit ThermoFisher Scientific Q33230DNA ShearingMegaruptor 3 System Diagenode B06010003Megaruptor 3 Shearing Kit Diagenode E07010003SMRTbell Library PreparationSMRTbell® Express Template Prep Kit 2.0 PacBio 100-938-900AMPure® PB Beads PacBio 100-265-900SMRTbell® Enzyme Clean Up Kit 2.0 (New*) PacBio 101-932-600Sequencing Primer v5 (New*) PacBio 102-067-400100% Ethanol, Molecular Biology Grade Any MLSWide Orifice Tips (Tips LTS W-O 200UL Fltr RT-L200WFLR) Rainin 30389241Lo-Bind 0.2 mL tube strips USA Scientific, TempAssure1402-4708Multi-channel Pipette Rainin, 17013810Magnetic separation rack V&P Scientific, Inc, VP 772F4-1Thermal Cycler that is 100 µL and 8-tube strip compatible Any MLSSize-selection (One of the following systems)PippinHT System Sage Science HTP00010.75% Agarose Gel Cassettes, Marker 75E Sage Science HPE7510BluePippin System Sage Science BLU00010.75% Agarose Cassettes, Marker S1 Sage Sciences BLF7510SageELF System Sage Science ELF00010.75% Agarose Cassettes Sage Science ELD7510SequencingSequel® II Binding Kit 2.2 (New*)PacBio 101-894-200Sequel® II Sequencing Kit 2.0 PacBio 101-820-200SMRT® Cell 8M Tray PacBio 101-389-001* To obtain a copy of the previous version of this Procedure & Checklist that specifies use of SMRTbell Enzyme Clean Up Kit (PN 101-746-400) and Sequencing Primer v2 (PN 101-847-900), contact ****************.HiFi Library Construction WorkflowPacBio recommends that gDNA samples be resuspended in an appropriate buffer (e.g., Qiagen Elution Buffer) before proceeding with DNA shearing.Figure 1: Workflow for preparing HiFi libraries using the SMRTbell Express Template Prep Kit 2.0.Reagent HandlingSeveral reagents in the SMRTbell Express Template Prep Kit 2.0 (shown in Table 2 below) are sensitive to temperature and vortexing. We recommend to:•Never leave reagents at room temperature.•Always work on ice when preparing master mixes.•Finger-tap followed by a quick spin prior to use.Reagent Where UsedDNA Prep Additive Remove single-strand overhangsDNA Prep Enzyme Remove single-strand overhangs DNA Damage Repair Mix v2 DNA Damage RepairEnd Prep Mix End-Repair/A-tailingOverhang Adapter v3 LigationLigation Mix LigationLigation Additive LigationLigation Enhancer LigationSMRTbell Enzyme Clean Up Mix Nuclease TreatmentSMRTbell Enzyme Cleanup Buffer 2.0 Nuclease TreatmentTable 2: Temperature sensitive reagentsGenomic DNA (gDNA) Quality EvaluationThis procedure requires high-quality, high-molecular weight input gDNA with a majority of the DNA fragments >50 kb as determined by pulsed-field gel or capillary electrophoresis. Any of the three commercially available systems listed in Table 4 below may be used to evaluate gDNA quality, but the Femto Pulse system is highly recommended for high-throughput library construction due to its ability to rapidly process multiple samples in a single run using very low amounts (<1 ng) of DNA per sample. Links to recommended procedures for each system are also provided in the table. Examples of gDNA quality assessment using Bio-Rad’s CHEF Mapper (2A) and Agilent Technologies’ Femto Pulse (2B) are shown in Figure 2. Lanes A3 and B1 correspond to high-quality gDNA samples that are suitable for HiFi library construction using this procedure. Lanes A4 and B2 show degraded gDNA samples that not suitable for use in this procedure.Method ProcedureFemto Pulse Agilent Technologies, Inc.Bio-Rad CHEF Mapper XA Pulsed Field Electrophoresis System Procedure & Checklist - Using the BIO-RAD® CHEF Mapper® XA Pulsed Field Electrophoresis SystemSage Science Pippin Pulse Procedure & Checklist - Using the Sage Science PippinPulse Electrophoresis Power Supply SystemTable 3. gDNA Quality Evaluation Methods and Procedures.Figure 2: Evaluation of high-molecular weight gDNA quality using two DNA sizing analysis systems. A) Bio-Rad CHEF Mapper and B) Agilent Technologies’ Femto Pulse.165.510 kb 50 kb 42 kb33 kb 21 kb 17.7 kb 1.3 kb1 bpLane 1: 8 kb - 48 kb Ladder (Bio-Rad) Lane 2: 5 kb ladder (Bio-Rad) Lane 3: HMW gDNA Lane 4: Degraded gDNALane 1: HMW gDNALane 2: Degraded gDNA Lane 3: 165 kb ladder48 kb-20 kb-80 kb----------10 kb-14322 1 3ABDNA Requirements for ShearingBefore shearing, ensure that the genomic DNA is in an appropriate buffer (e.g.,Qiagen Elution Buffer, 10 mM Tris-Cl, pH 8.5 or PacBio EB buffer). If you are unsure of the buffer composition or if the gDNA is not in Elution Buffer, perform a 1X AMPure PB bead purification followed by elution with Elution Buffer or an equivalent low salt buffer (i.e., 10 mM Tris-Cl, pH 8.5- 9.0).PacBio highly recommends Diagenode’s Megaruptor 3 system for shearing gDNA. The Megaruptor 3 system allows up to 8 gDNA samples to be processed simultaneously with a consistent fragment size distribution across multiple hydropore-syringes. Furthermore, the Megaruptor 3 system generates a narrower size distribution than the g-TUBE device (Covaris).Shearing Using Diagenode’s Megaruptor 3 SystemTo maximize HiFi yield per SMRT Cell, PacBio recommends fragmenting the gDNA to a size distribution mode between 15 kb – 18 kb for human whole genome sequencing. Libraries with a size distribution mode larger than 20 kb are not recommended for HiFi sequencing. Recommended library insert size distributions to use for different WGS applications are summarized in Table 4 below.Application Recommended Library Insert SizeHuman Variant Detection 15 – 18 kbHuman de Novo15 – 18 kbPlant/Animal de Novo15 – 20 kbTable 4: Library size recommendations for Human variant detection and de novo assembly.To shear gDNA on the Megaruptor 3 system, use a two-cycle shear method, which requires running a second round of shearing immediately following the first fragmentation step in the same hydropore-syringe. The recommended concentration is 83.3 ng/µL (5 µg of input DNA in 60 µL Elution Buffer).The DNA shearing guidelines below have been tested by PacBio on the Megaruptor 3 system only. The response of individual gDNA samples to the shearing recommendations described below may differ; therefore, performing a small-scale test shear is highly recommended, including the Megaruptor 3 system.For the Megaruptor and Megaruptor 2 systems, shearing optimization is necessary before proceeding with this Procedure & Checklist. The shearing procedure described in the “Shearing Using Diagenode’s Megaruptor 3 system” section below is not compatible with the Megaruptor or Megaruptor 2 systems. For Megaruptor and Megaruptor 2 systems, follow Diagenode’s DNA shearing recommendations described in their manual. For additional guidance, contact Technical Support or your local FAS.The g-TUBE device generates a broader DNA fragment size-distribution compared to the Megaruptor 3 system. Note that HiFi read quality and overall HiFi data yield may be reduced due to the residual presence of large DNA fragments generated by g-TUBEs. For additional guidance, contact Technical Support or your local FAS.Figure 3: Examples of human genomic DNA samples sheared to a target 15 kb - 18 kb size distribution mode using a 2-cycle shear method on the Megaruptor 3 system.Prepare SMRTbell LibrariesAlways work on ice throughout the library construction process. To process multiple samples at a time, the following equipment are required:• Lo-Bind tube strips• Multi-channel pipette• Wide-bore tips• Magnetic rack compatible with tube strips• Thermocycler compatible with tube stripsRemove Single-Strand OverhangsThe sample volume recovered from the Megaruptor 3 system after shearing is used directly in the single-strand overhang digestion step. Before proceeding, ensure that the sheared DNA is in Elution Buffer or an equivalent low salt buffer (i.e., 10 mM Tris-Cl, pH 8.5- 9.0). In this step, DNA Prep Additive is diluted first followed by digestion. Scale up the reaction volumes for digestion if working with multiple samples.1. Prepare the DNA Prep Additive. The DNA Prep Additive is diluted with Enzyme Dilution Buffer toa total volume of 5 µL. This amount is sufficient for processing 1 to 4 samples. The volume maynot be sufficient for 5 samples due to pipetting errors. We recommend scaling up the dilutionvolume based on the number of samples to be processed (example: prepare 2X volume for 8samples and 4X volume for 16 samples).Note: The diluted DNA Prep Additive should be used immediately and should not be stored.2. Prepare the digestion by following the reaction table below. For multiple samples, prepare amaster mix, followed by addition of 10.0μL master mix to each sheared DNA sample.3. Add 10.0 µL of the above master mix to the tube-strips containing 45.0 µL - 53.0 µL of shearedDNA. The total volume in this step is 55.0 µL - 63.0 µL.4. Using a multi-channel pipette, mix the reaction wells by pipetting up and down 10 times with wide-orifice pipette tips.5. Spin down the contents of the tube strips with a quick spin in a microfuge.6. Incubate at 37°C for 15 minutes, then return the reaction to 4°C.7. Proceed to the next step.Repair DNA DamageTo each Reaction Mix 1, add 2.0 µL of DNA Damage Repair Mix v2.1. Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips.2. Spin down the contents of the tube strips with a quick spin in a microfuge.3. Incubate at 37°C for 30 minutes, then return the reaction to 4°C.4. Proceed to the next step.End-Repair/A-tailingTo each Reaction Mix 2, add 3.0 µL of End Prep Mix.1. Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips.2. Spin down the contents of the tube strips with a quick spin in a microfuge.3. Incubate at 20°C for 10 minutes.4. Incubate at 65°C for 30 minutes, then return the reaction to 4°C.5. Proceed to the next step.Adapter LigationIn this step, 5.0 µL of Overhang Adapter is added to each Reaction Mix 3 (from the previous step). Then, 32.0 µL of the ligase master mix is added to each Reaction Mix 3/Adapter Mix for incubation. Always work on ice. 1. To each Reaction Mix 3, add 5.0 µL of Overhang Adapter.2. Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips. Leave the tube strips on ice.3. Prepare a Master Mix containing Ligation Enhancer, Ligation Additive and Ligation Mix using the table4. Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips. It is important to mixwell.5. To the Reaction Mix 3/Adapter Mix, add 32.0 µL of the Ligase Master Mix. The total volume in this step is97.0 µL- 105.0 µL.6. Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips. It is important to mixwell.7. Incubate at 20°C for 1 hour. Optional: The Ligation reaction may also be left at 20°C overnight.8. Proceed to the next step.Purify SMRTbell Library Using 1.0X AMPure® PB BeadsPage 11 PN 101-853-100 Version 05(August 2021)Nuclease Treatment of SMRTbell LibraryTo each library sample, add the nuclease mix to remove damaged SMRTbell templates.1. Prepare a Master Mix of the Enzyme Cleanup Mix and Buffer.2. Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips. It is important to mixwell.3. Spin down the contents of the tube strips with a quick spin in a microfuge.4. To each 15.0μL of sample, add 55.0 μL of Nuclease Master Mix. The total reaction volume at this step is70.0 µL.5. Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips. It is important to mixwell.6. Incubate at 37°C for 30 mins and store on ice immediately.7. Spin down the contents of tube strips with a quick spin in a microfuge.8. Proceed directly to the AMPure PB bead purification step below immediately. Do not store samples at thisstage. Do not let samples sit for long periods of time. Always work on ice.Page 12 PN 101-853-100 Version 05(August 2021)Purify SMRTbell Library Using 1.0X AMPure® PB BeadsSize Selection of SMRTbell LibrariesFor high-throughput whole genome sequencing applications, PacBio highly recommends the PippinHT system (Sage Science) for size-selection of SMRTbell libraries for HiFi sequencing. Typical recovery yields are 35% - 50% and are highly dependent on the size distribution of the starting SMRTbell library.Size Selection Using the PippinHT SystemVerify that your PippinHT system software is up to date and follow the procedure below to remove SMRTbellSize Selection Using the BluePippin SystemSage Science’s BluePippin system may also be used for size-selection of HiFi SMRTbell libraries. Verify that your BluePippin system software is up to date and follow the procedure below to remove SMRTbell templates <10 kb using the BluePippin system. Typical recovery yields are highly dependent on the size distribution of the starting SMRTbell library. For the latest BluePippin system User Manual and guidance on size-selection protocols, contact Sage Science ().Size Selection Using the SageELF SystemSage Science’s SageELF system may also be used to fractionate SMRTbell libraries for HiFi whole genome sequencing applications. Verify that your SageELF system software is up to date and follow the size selection procedure below. For the latest SageELF User Manual and guidance on size-selection protocols, contact Sage Science ().6Set up the run Protocol:– In the “Protocol Editor” tab, click on the “New Protocol” button.– Select the “0.75% 1-18kb v2” in the cassette definition menu.– Select “size-based” for separation mode.– Enter 3450 in the “Target Value” field and move the bar slider to selectwell #12.– Save as new protocol.– On the Main screen, clear previous run data, select cassette description,cassette definition and protocol, enter sample ID(s).– Select in the Nest Selector the cartridge that will be run.7Start the run.8 Once the run is complete, (approximately 4.5 hours), collect 30 μL of the respectivefractions from the elution wells. Fractions of interest are typically ~11 kb, ~13 kb,~15 kb, ~17 kb.9 Check the sizes of all 12 fractions by loading on a Femto Pulse. To determine theaverage library size, perform a smear analysis by selecting the region of interestby defining the start and end points of the fractions.10 Pool together fractions that have an average library size 10 – 20 kb.11 Proceed to the AMPure PB Bead purification step.Purify Size-Selected HiFi Library Fractions with 1.0X AMPure ® PB BeadsSequencing PreparationSee Quick Reference Card - Loading and Pre-Extension Recommendations for Sequel II/IIe Systems .For Research Use Only. Not for use in diagnostic procedures. © Copyright 2020 - 2021, Pacific Biosciences of California, Inc. All rights reserved. Information in thisdocument is subject to change without notice. Pacific Biosciences assumes no responsibility for any errors or omissions in this document. Certain notices, terms, conditions and/o r use restrictions may pertain to your use of Pacific Biosciences products and/or third p arty products. Please refer to the applicable PacificBiosciences Terms and Conditions of S a le and to the applicable license terms at /lice nses.html. Pacific Biosciences, the Pacific Biosciences logo, PacBio, S M RT, SMRTbell, Iso-Seq and Sequel are trademarks of Pacific Biosciences. Femto Pulse and Fragment Analyzer are trademarks of Agilent Technologies. All other trademarks are the sole property of their respective owners.Revision History (Description)Version Date Initial release.01 September 2019 Internal revision with no content change (not uploaded to website).02 December 2019 On page 1, changed “HiFi reads” to just “Reads”. On page 12, under Repair DNA Damage,corrected “remove single strand overhangs” to “repair DNA damage”. On page 13, corrected “remove single strand overhangs” to “adapter ligation”.03 January 2020 Updated for SMRTbell Enzyme Clean Up Kit 2.0 and Sequencing Primer v5.04 April 2021 Removed SMRT Link Sample Setup and Run Design tables. Added reference to QRC.05August 2021。

Takara Bio USA, Inc. 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: *******************************United States/Canada Asia Pacific Europe Japan Page 1 of 9Takara Bio USA, Inc. Guide-it™ Recombinant Cas9 (3 µg/µl) User ManualCat. Nos. 632640, 632641 (021121)Table of ContentsI. Introduction (3)A. Summary (3)II. List of Components (3)III. Additional Materials Required (3)A. Electroporation Supplies (3)B. Mammalian Cell Culture Supplies (4)C. General Supplies (4)D. sgRNA Development and Production (4)E. Detection and Characterization of Gene Editing (4)IV. Protocol Overview (5)V. Electroporation Protocol for Neon Transfection System (5)A. Protocol: Preparation of Cells and Media (5)B. Protocol: Preparation of Cas9-sgRNA RNP Complex (6)C. Protocol: Electroporation (6)VI. Electroporation Protocol for 4D-Nucleofector System (7)A. Protocol: Preparation of Cells (7)B. Protocol: Preparation of Cas9-sgRNA RNP Complex (8)C. Protocol: Electroporation (8)VII. References (9)Table of FiguresFigure 1. Protocol overview for Guide-it Recombinant Cas9 (3 µg/µl). (5)I. IntroductionA. SummaryThe CRISPR/Cas9 system has emerged as a powerful tool for gene editing because of its high targetingspecificity, editing efficiency, and ease of use in virtually any organism. CRISPR/Cas9 technologyconsists of two key components that form a complex: Cas9 endonuclease and a single guide RNA(sgRNA) that directs Cas9 to cleave genomic DNA in a sequence-specific manner (Jinek et al. 2012).This RNA-programmable method exploits the error-prone nature of the non-homologous end joiningDNA repair pathway (NHEJ) to generate gene knockouts (via insertion/deletion). The method can also beused to generate knockins via the homology-directed repair (HDR) pathway.CRISPR/Cas9 system components have been delivered successfully into target cells through a variety ofapproaches, including vector-based expression systems, transfection of RNA, and more recently,introduction of Cas9-sgRNA ribonucleoprotein (RNP) complexes. Delivery of Cas9-sgRNA RNPsprovides a fast turnaround for gene-editing experiments while minimizing the likelihood of off-targeteffects compared to vector-based approaches (Sander and Joung 2014), and this approach has beenoptimized for various cell types using microinjection, electroporation, and lipid-mediated transfection(Liang et al. 2015).Guide-it Recombinant Cas9 (3 µg/µl) is a recombinant wild-type Streptococcus pyogenes Cas9 nucleaseexpressed with a C-terminal nuclear-localization signal (NLS)and purified from E. coli for use inCRISPR/Cas9-mediated gene editing experiments. The Cas9 protein solution has been verified to besterile and well-tolerated by mammalian cells when electroporated as a ribonucleoprotein complex (RNP)with a single guide RNA (sgRNA) for knockout experiments, or as an RNP with a donor repair templatefor knockin experiments.II. List of ComponentsGuide-it Recombinant Cas9 (3 µg/µl) (Cat. No. 632641)•100 µg Guide-it Recombinant Cas9 (3 µg/µl)Guide-it Recombinant Cas9 (3 µg/µl) (Cat. No. 632640)• 3 x 100 µg Guide-it Recombinant Cas9 (3 µg/µl)•Store Guide-it Recombinant Cas9 (3 µg/µl) at –70°C.•Avoid repeated freeze/thaw cycles. We recommend preparing aliquots upon initial thawing of Guide-it Recombinant Cas9 (3 µg/µl).III. Additional Materials RequiredThe following reagents/materials are required but not included.A. Electroporation SuppliesUse of this product requires an electroporator, electroporation chamber (typically cuvettes or tips), and anelectroporation buffer that is suitable for your target cells. Here we provide separate guidelines for theNeon Transfection System (Thermo Fisher Scientific, Cat. No. MPK5000) and the 4D-NucleofectorSystem (Lonza, Cat. No. AAF-1002B).B. Mammalian Cell Culture Supplies•Culture medium, supplies, and additives specific to your target cells•Cell culture plates•PBS without Ca2+ or Mg2+•Trypsin/EDTA or equivalent•Humidified incubator (set at 37º C, 5% CO2)C. General Supplies•Single-channel pipettes•Nuclease-free thin-wall PCR tubes or stripsD. sgRNA Development and ProductionCRISPR/Cas9 gene editing requires a custom sgRNA with a user-designed targeting sequence that ishomologous to the target gene or genomic region of interest. Selecting an appropriate DNA sequence at the target region is critical for maximizing the potential for efficient cleavage at the target site and forminimizing the likelihood of non-specific cleavage events. There are several freely available online tools that can be helpful for determining suitable sgRNA target sequences for a given organism and genomic target. For a list of these tools, please refer to:/US/Products/Genome_Editing/CRISPR_Cas9/Resources/Online_tools_for_gui de_RNA_design.NOTE: For many applications, it is advisable to design and test several variant sgRNAs against the same genomic target region.Candidate sgRNAs must ultimately be produced in sufficient quantity for the generation of functionalCas9-sgRNA RNPs. For development and production of user-designed sgRNAs, we recommend either of the following kits:•For constructing and purifying sgRNAs: Guide-it sgRNA In Vitro Transcription Kit (Takara Bio, Cat.No. 632635).•For constructing and purifying sgRNAs, and testing target cleavage efficiencies in vitro: Guide-it Complete sgRNA Screening System (Takara Bio, Cat. No. 632636).E. Detection and Characterization of Gene EditingThese items are recommended for determining the efficiency of gene editing and the nature of the edits:Cat. No. Product Size631443 Guide-it Mutation Detection Kit 100 rxns631448 Guide-it Mutation Detection Kit 25 rxns632611 Guide-it Genotype Confirmation Kit 100 rxns631444 Guide-it Indel Identification Kit 10 rxnsIV. Protocol OverviewPlease read each relevant protocol completely before starting. Successful results depend on understanding and performing the following steps correctly.Figure 1. Protocol overview for Guide-it Recombinant Cas9 (3 µg/µl).V. Electroporation Protocol for Neon Transfection SystemHere we provide protocols for performing knockout and knockin experiments in hiPS cells and CD34-positive stem cells using the Neon Transfection System. While these protocols may serve as a helpful starting point forelectroporation of other cell types as well, further optimization will be required. Please refer to the Neon Transfection System User Manual and manufacturer’s website for detailed operating instructions for the Neon Transfection System.A. Protocol: Preparation of Cells and MediaCultured target cells are harvested, washed, and resuspended in the appropriate buffer.1.Prepare a sufficient number of fresh cells for your experiment.NOTE: Each electroporation requires 1 x 105 cells. However, due to the potential variation of pipetteand tip volumes, we recommend preparing 1.5X the necessary volume of cell suspension (i.e., 1.5 x 105cells) for electroporation with a 10-µl Neon Tip to ensure that there is sufficient volume.2.For hiPS cells (adherent cells), continue to Step3. For CD34-positive stem cells (suspension cells),skip to Step 5.3.Aspirate the medium, wash the cell layer once with PBS (without Ca2+ and Mg2+), and dissociate thecells using TrypLE Select Enzyme (1X) (Thermo Fisher Scientific, Cat. No. 12563011).4.Harvest the cells in growth medium.5.Take an aliquot of the cell suspension and measure the cell density using your preferred method.6.Harvest the cells by centrifugation at 400g for 5 min in a 15-ml conical tube.7.Wash the cells once with PBS (without Ca2+ and Mg2+), and then resuspend hiPS cells in Buffer R andCD34-positive stem cells in Buffer T (included with Neon kits) at a concentration of 2 x 107 cells/ml(i.e., 1.5 x 105 cells in 7.5 µl).NOTE: Use Resuspension Buffer R for established adherent and suspension cells as well as primaryadherent cells, and use Resuspension Buffer T for primary blood-derived suspension cells.8.Keep the cell suspension on ice until use.B. Protocol: Preparation of Cas9-sgRNA RNP ComplexCas9 and sgRNA components are combined to form RNP complexes for electroporation.1.Thaw Guide-it Recombinant Cas9 (3 µg/µl) and sgRNA solutions at room temperature.NOTE: We recommend preparing aliquots upon initial thawing of Guide-it Recombinant Cas9(3 µg/µl) to avoid repeated freeze/thaw cycles.bine the following components in a 200-µl PCR tube to mix the Cas9 protein and sgRNA at a 5:1mass ratio. The molar ratio of Cas9 protein to sgRNA will be approximately 1:1 in this mixture, andthe total volume will be 7.5 µl. Be sure to use the same buffer that was used to resuspend the cells.NOTE: The reaction volume indicated below is 1.5X the required volume.Per reaction:0.45 μl* sgRNA (e.g., 1 µg/µl)0.75 μl Guide-it Recombinant Cas9 (3 µg/µl)6.3 μl* Resuspension Buffer R or T7.5 μl Total volume*The added volume of sgRNA will vary depending on sgRNA concentration, and the added volume ofResuspension Buffer should be adjusted such that the total reaction volume is 7.5 µl. The volumesindicated above are based on a sgRNA concentration of 1 µg/µl.NOTE: Make a master mix if you are performing multiple electroporations.NOTE: The optimal amount of RNP complex may vary for different cell types.NOTE: To maximize electroporation efficiency, the combined volume of the Cas9 and sgRNA solutionsshould be ≤20% of the total volume of the Cas9-sgRNA RNP complex reaction (e.g., for the 7.5-µlreaction specified above, the combined volume of the sgRNA and Cas9 solutions should be ≤1.5 µl).NOTE: If you plan to use donor DNA to induce HDR-mediated knockin, add the DNA after thesubsequent incubation step (Step 3). We recommend using ≤1 µg of DNA for knockin experiments.Adjust the volume of Resuspension Buffer R or T included in the reaction such that the final volumeupon addition of donor DNA is 7.5 µl.3.Mix the reaction well by gently pipetting up and down. Incubate using a thermal cycler preheated to37°C with the following program:37°C 5 min4°C hold4.OPTIONAL: Add donor DNA and keep on ice until use.C. Protocol: ElectroporationCas9-sgRNA RNPs are electroporated into target cells.1.Fill the Neon Tube with 3 ml of Buffer E (included with Neon kits) and insert the Neon Tube into theNeon Pipette Station.ing the touchscreen on the Neon system, set up the electroporation parameters as follows:Pulse voltage / Pulse width / Pulse number = 1100 v / 20 ms / 2 pulsesNOTE: We have used these parameters successfully for hiPS cells and CD34-positive stem cellsusing the Neon Transfection System. Optimization of electroporation parameters will be required fordifferent target cell types. Suggested parameters for different cell types are included in thesupplementary material for (Liang et al. 2015).3.Gently resuspend the cells by tapping, and transfer 7.5 µl of the cell suspension into the PCR tubecontaining the 7.5 µl of Cas9-sgRNA RNP complex solution.4.Mix well by gently pipetting up and down.5.Insert the Neon Pipette into the Neon Tip and confirm that the pipette and tip are tightly connected.ing the Neon Pipette, aspirate the mixture slowly into the Neon Tip.NOTE: Avoid any air bubbles in the tip. If you notice air bubbles, place the sample back into thePCR tube and aspirate again into the tip without any air bubbles.7.Insert the Neon Pipette into the Neon Tube placed in the Neon Pipette Station and run the program.8.Remove the pipette very carefully and transfer the cells into a cell culture plate with pre-warmedmedium.NOTE: Use an appropriate well plate for your target-cell type. We had success using 24 and 48-wellplates for CD34-positive stem cells and hiPS cells, respectively. hiPS cells typically require greaterconfluence than regular adherent cells.9.Shake the plate appropriately to disperse the cells and incubate at 37°C in a humidified incubator with5% CO2 until the next necessary procedure.VI. Electroporation Protocol for 4D-Nucleofector SystemHere we provide protocols for performing knockout and knockin experiments in Jurkat and CD34-positive stem cells using the 4D-Nucleofector System with 16-well Nucleocuvette Strips. While these protocols may serve as a helpful starting point for electroporation of other cell types as well, further optimization will be required. Please refer to the 4D-Nucleofector System User Manual and manufacturer’s website for more detailed information.A. Protocol: Preparation of CellsCultured target cells are harvested, washed, and resuspended in the appropriate solution.1.Prepare a sufficient number of fresh cells for your experiment.NOTE: Each electroporation requires 2 x 105 cells.2.Take an aliquot of the cell suspension and measure the cell density using your preferred method.3.Harvest the cells by centrifugation at 400g for 5 min in a 15-ml conical tube.4.Wash once with PBS (without Ca2+ and Mg2+), and then resuspend Jurkat cells in SE NucleofectorSolution (with supplement) and CD34-positive stem cells in P3 Nucleofector Solution (withsupplement) at a concentration of 1 x 107 cells/ml (i.e., 2 x 105 cells in 20 µl).NOTE: Please refer to the 4D-Nucleofector System User Manual and manufacturer’s website formore information about working with other cell types.NOTE: 20 µl of cell suspension will be needed per well of the Nucleocuvette Strip.5.Keep the cell suspension on ice until use.B. Protocol: Preparation of Cas9-sgRNA RNP ComplexCas9 and sgRNA components are combined to form RNP complexes for electroporation.1.Thaw Guide-it Recombinant Cas9 (3 µg/µl) and sgRNA solutions at room temperature.NOTE: We recommend preparing aliquots upon initial thawing of Guide-it Recombinant Cas9(3 µg/µl) to avoid repeated freeze/thaw cycles.bine the following components in a 200-µl PCR tube to mix the Cas9 protein and sgRNA at a 5:1mass ratio:Per reaction (e.g. 5 µl):2μl* sgRNA (e.g., 1 µg/µl)3.3 μl Guide-it Recombinant Cas9 (3 µg/µl)5.3 μl Total volume*The added volume of sgRNA will vary depending on sgRNA concentration. The volume indicated aboveis based on an sgRNA concentration of 1 µg/µl.NOTE: Make a master mix if you are performing multiple electroporations.NOTE: The RNP volume required for efficient transfection needs to be optimized for different celltypes. Usually ≤10 µl of the RNP solution will be tested for electroporation in each well of the 16-well Nucleocuvette Strip.3.Mix the reaction well by gently pipetting up and down. Incubate using a thermal cycler preheated to37°C with the following program:37°C 5 min4°C HoldC. Protocol: ElectroporationCas9-sgRNA RNPs are electroporated into target cells.bel wells of Nucleocuvette Strips to be used for electroporation.bine 20 µl of cell suspension with 5 µl Cas9-sgRNA RNP complex solution in each well of theNucleocuvette Strip, and mix well by gently pipetting up and down.NOTE: If you plan to use donor DNA to induce HDR-mediated knockin, add the donor DNA at thisstep.3.Select the program CL-120 for Jurkat cells or the program D0-100 for CD34-positive stem cells.4.Insert the Nucleocuvette Strip into the Nucleofector machine and run the program.5.Add 80 µl of pre-warmed medium to each cuvette and allow electroporated cells to recover for 12min post-transfection at room temperature.6.Gently collect the cells along with the media from each well and transfer to individual wells of a 48-well plate containing pre-warmed medium.7.Shake the plate appropriately to disperse the cells and incubate at 37°C in a humidified incubator with5% CO2 until the next necessary procedure.VII. References1.Jinek, M., Chylinsky, K., Fonfara, I., Hauer, M., Doudna, J.A., & Charpentier, E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337(6096), 816–21 (2012).2.Liang, X. et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J.Biotechnol.208, 44–53 (2015).3.Sander, J.D. & Joung, J.K. CRISPR-Cas9 systems for genomic editing, regulation and targeting. Nat.Biotechnol. 32, 347-55 (2014).Contact UsCustomer Service/Ordering Technical Supporttel: 800.662.2566 (toll-free) tel: 800.662.2566 (toll-free)fax: 800.424.1350 (toll-free) fax: 800.424.1350 (toll-free)web: web: e-mail: **********************e-mail: ********************Notice to PurchaserOur products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.Your use of this product is also subject to compliance with any applicable licensing requirements described on the product’s web page at . It is your responsibility to review, understand and adhere to any restrictions imposed by such statements© 2021 Takara Bio Inc. All Rights Reserved.All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at .This document has been reviewed and approved by the Quality Department.。

SilkTest® 2009 R2 Quick Start Tutorial for the SilkTest RecorderBorland Software Corporation4 Hutton Centre Dr., Suite 900Santa Ana, CA 92707Copyright 2009 Micro Focus (IP) Limited. All Rights Reserved. SilkTest contains derivative worksof Borland Software Corporation, Copyright 2009 Borland Software Corporation (a Micro Focuscompany).MICRO FOCUS and the Micro Focus logo, among others, are trademarks or registered trademarksof Micro Focus (IP) Limited or its subsidiaries or affiliated companies in the United States, UnitedKingdom and other countries.BORLAND, the Borland logo and SilkTest are trademarks or registered trademarks of BorlandSoftware Corporation or its subsidiaries or affiliated companies in the United States, UnitedKingdom and other countries.All other marks are the property of their respective owners.iiContentsQuick Start Tutorial for the SilkTest Recorder (4)Starting the SilkT est Recorder (4)Recording a T est Case for the Insurance Company Web Site (4)Replaying the T est Case for the Insurance Company Web Site (5)Exporting a T est Case or Project (6)Exporting a Project to Silk4J (6)Exporting a T est Method to Silk4J (6)Exporting a Project to SilkT est (7)Exporting a T est Case to SilkT est (8)Modifying the T est Case for the Insurance Company Web Site (8)Contents | 3Quick Start Tutorial for the SilkTest Recorder This tutorial provides a step-by-step introduction to using the SilkT est Recorder to test a Web site.The Recorderuses dynamic object recognition to record and replay test cases that use XPath queries to find and identifyobjects.The Recorder can test the following application types:•Adobe Flex•Java SWT/RCP•Windows API-based client/server•Windows Forms•xBrowser (Web applications)For the sake of simplicity, this guide assumes that you have installed the Recorder and are using the InsuranceCompany sample Web site, /InsuranceWebExtJS/.For additional information about Recorder, refer to the Recorder Help.To view the help, open Recorder andthen choose Help➤Help Contents.Starting the SilkTest RecorderChoose Start➤Programs➤Silk➤SilkTest <version>➤SilkTest Recorder.Recorder opens and the SilkTest Open Agent icon appears in the system tray.Recording a Test Case for the Insurance Company Web Site Record a test case for the Insurance Company sample Web site to see how the Recorder creates a test.1.Perform one of the following steps:•Click in the toolbar.•Choose Record➤Start Recording.The New Application Configuration wizard opens.2.Double-click Web Site Test Configuration.The New Web Site Configuration page opens.3.From the Browser Type list box, select Internet Explorer.Y ou can use Firefox to replay tests but not to record them.4.Perform one of the following steps:•Use existing browser– Click this option button to use a browser that is already open when you configure the test. For example, if the Web page that you want to test is already displayed in a browser, you mightwant to use this option.•Start new browser– Click this option button to start a new browser instance when you configure the test.Then, in the Browse to URL text box specify the Web page to open.4 | Quick Start Tutorial for the SilkTest RecorderFor this tutorial, close all open browsers and then click Start new browser and specify/InsuranceWebExtJS/.5.Click Finish.The Web site opens.The Recorder creates a base state and starts recording.6.In the Insurance Company Web site, perform the following steps:a)From the Select a Service or login list box, select Auto Quote.The Automobile Instant Quote page opens.b)Type a zip code and email address in the appropriate text boxes, click an automobile type, and thenclick Next.c)Specify an age, click a gender and driving record type, and then click Next.d)Specify a year, make, and model, click the financial info type, and then click Next.A summary of the information you specified appears.e)Point to the Zip Code that you specified and press Ctrl+Alt to add a verification to the script.Y ou can add a verification for any of the information that appears.The Verify Properties dialog box opens.f)Check the textContents check box and then click OK.A verification action is added to the script for the zip code text.An action that corresponds with each step is recorded.7.In the Recorder, perform one of the following steps:•Click in the toolbar.•Choose Record➤Stop Recording.8.Choose File➤Save.a)Navigate to the location in which you want to save the test.b)In the File name text box, type the name for the test and then click Save.For example, type ZipTest.Replay the test to ensure that it works as expected.Y ou can modify the test to make changes if necessary. Replaying the Test Case for the Insurance Company WebSiteReplay a test to ensure that it works as expected.1.On the main window, from the Replay speed list box, select the speed to use to replay the test.•Fast– This option is the fastest choice for test replay and the true speed at which scripts are executed.The other speeds use a delay mechanism, so you can see the test run.•Medium– In most cases, this option enables you to see each action.•Slow– T o ensure that you see each action, choose this option.Y ou can determine the speed of the test by checking the Replay duration time posted next the Replayspeed list box.2.To replay the entire test, click in the toolbar or choose Replay➤Replay All.The Recorder plays back the test.3.If an error occurs, perform one of the following steps:Quick Start Tutorial for the SilkTest Recorder | 5Click Retry to replay the current action.••Click Stop to end the test.•Click Skip to advance to the next action in the test.Export the test to use it with SilkT est or Silk4J. Or, modify the test to make changes if necessary.Exporting a Test Case or ProjectUse the following procedures to export a test case or project to SilkTest or to Silk4J.Exporting a Project to Silk4JExport projects to use Silk4J as the primary GUI for tests and to organize test methods.1.Choose File➤Export.The Export wizard opens.2.Double-click Export as Silk4J Project.The Export as Silk4J Project page opens.3.In the Project location text box, specify the location to which to export the project.Optional: Click and navigate to the folder that you want to use.4.In the Project name text box, specify the project name.For example, type Web Sample Project.5.In the Package text box, specify the package name.For example, type com.example.6.In the Test class text box, specify the class name to which the test belongs.For example, type AutoTests.7.In the Test method text box, specify a name for the test method.For example, type testAutoInput.8.From the File encoding list box, click the type of file encoding to use.9.Click Finish.The Recorder creates a project and exports it to Silk4J.Import the project with Silk4J.The new project contains a base state and test method and is ready for testing.For details about importing a project, refer to the Silk4J User Guide.Exporting a Test Method to Silk4JExport test methods to use Silk4J as the primary GUI for tests or to copy the JTF script to the Clipboard.1.Choose File➤Export.The Export wizard opens.2.Double-click Export as JTF Script.The Export to JTF page opens.3.From the Export to list box, click one of the following options:6 | Quick Start Tutorial for the SilkTest RecorderClipboard– Copies the JTF script to the clipboard.Y ou might choose this option if you want to copy •and paste the script into an existing JTF script.•JTF Script– Exports the script to Silk4J.Y ou might choose this option if you want to create a new script or overwrite an existing JTF script.4.In the Test method text box, specify a name for the test method.For example, type testAutoInput.5.In the Package text box, specify the package name.For example, type com.example.6.In the Test class text box, specify the class name to which the test belongs.For example, type AutoTests.7.In the Source folder text box, specify the location to which to export the test.Optional: Click and navigate to the folder that you want to use.8.From the File encoding list box, click the type of file encoding to use.9.To include the base state in the exported script, check the Use base state check box.The base state makes sure that the application that you want to test is running and in the foreground.Thisensures that tests will always start with the same application state, which makes them more reliable. Inorder to use the base state, it is necessary to specify what the main window looks like and how to launchthe application that you want to test if it is not running. Creating a base state is optional. However, it isrecommended as a best practice.If you export to JTF, a separate silk4J.settings file is created for the base state. If you export to theClipboard, a Before method includes the base state.10.Click Finish.The Recorder creates a Java script and exports it to Silk4J or to the Clipboard.Exporting a Project to SilkTestExport projects to use SilkT est as the primary GUI, group test cases into projects, or to share data with other products such as SilkCentral T est Manager.1.Choose File➤Export.The Export wizard opens.2.Double-click Export as SilkTest Project.The Export as SilkTest Project page opens.3.In the Project location text box, specify the location to which to export the project.Optional: Click and navigate to the folder that you want to use.4.In the Project name text box, specify the project name.For example, type Web Sample Project.5.In the 4Test script text box, specify the script file name.For example, type AutoTests.t.Optional: Click and navigate to the folder that you want to use.6.In the Test case text box, specify a name for the test case.For example, type testAutoInput.Quick Start Tutorial for the SilkTest Recorder | 77.To start SilkTest after the test case is exported, check the Open the exported project in SilkTest checkbox.8.Click Finish.The Recorder creates a project that includes a script that uses the 4Test language and a recovery file andexports the project to SilkTest.Use SilkTest to work with the exported project.The new project contains a base state and test case and isready for testing.Exporting a Test Case to SilkTestExport test cases to use SilkT est as the primary GUI or to copy the script to the Clipboard.1.Choose File➤Export.The Export wizard opens.2.Double-click Export as 4Test Script.The Export to 4Test Script page opens.3.From the Export to list box, click one of the following options:•Clipboard– Copies the script to the clipboard.Y ou might choose this option if you want to copy and paste the script into an existing 4T est script.•4Test Script– Exports the script to SilkT est.Y ou might choose this option if you want to create a new script or overwrite an existing script.4.In the Test case text box, specify a name for the test case.For example, type testAutoInput.5.In the 4Test script text box, specify the script file name.For example, type AutoTests.t.Optional: Click and navigate to the folder that you want to use.6.To start SilkTest after the test case is exported, check the Open the exported script in SilkTest checkbox.7.Click Finish.The Recorder creates a script that uses the 4Test language and exports it to SilkT est or the Clipboard.Modifying the Test Case for the Insurance Company WebSiteAfter you record a test case, perform this step to manually modify the test. For example, you might add anadditional action, change the order of the recorded steps, or change the parameters for a specific action.1.Open the test that you want to modify.2.Navigate to the Web page on which the test ended in the initial recording.For instance, if the existing test includes multiple Web pages, navigate to the last page in the test.In this case, the existing test recording ended on the Automobile Instant Quote summary page.We wantto return to the Home page, which the base state will do automatically when we record.3.To record the modifications, perform the following steps:8 | Quick Start Tutorial for the SilkTest Recordera)Click in the toolbar.b)From the Select a Service or login list box, select Agent Lookup.The Find an Insurance Co. Agent page opens.c)Click in the toolbar.4.To change an existing action, parameter, or locator string, perform the following steps:a)Click the row that you want to change in the Actions grid.For example, click the row that contains the SetText action for the e-mail address parameter.The Action Details tab shows the Locator, Action, and Parameters.b)To change the locator, type a string in the Locator text box.The locator string identifies the object that you want to test.c)Click Validate Locator.The Recorder validates the new locator. If the string is not valid or the object cannot be found, an erroris displayed.d)To change the action, select an action from the Action list.e)To change the parameters, type a parameter value in the appropriate text box.For example, change the text parameter to "************************" for the SetText action.Any changes that you make display in the Actions grid immediately.5.To manually add an action, perform the following steps:Typically, you record actions that you want to add to a test. However, you can also manually add an actionby copying and pasting an existing action that you then modify.a)Click an action in the Actions list.Note:Y ou can insert multiple actions by pressing Ctrl or Shift and then clicking the actionsthat you want to copy.b)Perform one of the following steps:•Choose Edit➤Copy Selected Actions and then choose Edit➤Paste Actions.•Press Ctrl+C and then press Ctrl+V.A new action displays below the action that you selected.c)In the Action Details view, modify the action to meet your needs by changing the locator, action name,or parameters as needed.6.Choose File➤Save.Replay the test to ensure that it works as expected.Quick Start Tutorial for the SilkTest Recorder | 9IndexBbase staterun before record/replay8 Eexportingtest cases to Silk4J6test cases to SilkT est7, 8 Rrecordingadditional actions8recording (continued) test cases4 replaying test cases5 Sstarting Recorder4 Ttest casesexporting6, 7, 8modifying8recording410 | Index。

Bisulfite Sequencing1. General InformationAs the first discovered mark, DNA methylation plays a vital role in genome dynamics. It is implicated in repression of transcriptional activity and in animals it predominantly involves the addition of a methyl group to the carbon-5 position of residues at cytosine guanine dinucleotide (CpG) sites.Bisulfite sequencing is a powerful technique to study DNA methylation. Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. The following PCR amplification treatment converts uracil to thymine. Thus, bisulfite treatment introduces specific changes in the DNA sequence that depends on the methylation status of individual cytosine residues. Coupled with new-generation sequencing technology, it allows for an unbiased genome-wide analysis of DNA methylation and various analyses can be performed on the altered sequence. Bisulfite-seq is the golden standard for DNA methylation analysis. Process includes: treating of DNA with bisulfite to convert cytosine residues to uracil, while leaving 5-methylcytosine residues unaffected; running PCR to convert all the uracil to thymines; in the end, sequencing the PCR product and performingthe bioinformatics analysis compared with the untreated genome to profile quantitive regional methylation pattern.Compared to Sanger sequencing, Bisulfite sequencing is a low-cost method with high reliability and accuracy to determine each cytosine methylation state. Based on new-generation sequencing technology, it avoids mass work of clone sequencing and complicated process. The sequencing primers which will be added to the DNA fragments to process sequencing also can be treated as random amplication primer for DNA samples. Thus, primer designing is not necessary and work of PCR will be decreased when compared to Sanger sequencing to profile genome-wide DNA methylation pattern.2. Experimental PipelineBisulfite treatment of sample DNA and DNA sequencingPerform the bisulfite treatment of qualified DNA and forward to TA clone test. Then the DNA sequencing is carried out using on the new-generation sequencing technology.DNA was extracted and processed by bisulfite sodium and subjected to Illumina GA sequencing with methylated adaptor. The detailed pipeline is showed as following (Figure 6-2-1):3. Bioinformatics AnalysisThe bioinformatics analysis of Bisulfite sequencing is based on the SOAP alignment with C T mismatches tolerated. Ideally, this method would determine the methylation status separately for each allele, even each single strand. The methylation status for each allele is the most important information to detect differentially methylated region as the candidate of imprinting gene. The methylation status for each single strand could be used to describe thehemi-methylation (Figure 6-2-2) .3.1 Basic bioinformatics analysisData production statistics includes image recognition, base calling, filtering adapter sequences and detecting contaminants of sample.3.2 Advanced bioinformatics analysisMap bisulfite-seq reads to referenceGet the methylation profile of the mapped reads.Gather the methylation information of each base of the mapped reads.Get the methylation rate information of all methylated C in CpG of each chromosome.Get the methylation rate information of all dispersed C of each chromosome. Provide the methylated CpG information in different gene regions.Provide the methylated dispersed C information in different gene regions. Provide the methylated CpG information of genome sequence with different features.Provide the methylated dispersed C information of genome sequence with different features.4. Case Studies4.1 Study on an individual human genomeThe match information of all the base sites is gathered and decoded after SOAP alignment. At each base site, tags number which suggests this site to be methylated or not will be given. In the following analysis, the C bases with copy number > 1.5 and quality < 14 are filtered out. The methylated rate of CpG or non-CpG is based on tags number which supports this site to be methylated comparing with and number of all effective tags.5. Frequently Asked Questions1) How many nucleotides are required for Bisulfite sequencing analysis?In order to ensure at least 10X coverage of genome size,we suggest 10 g of DNA to be provided as the mininum amount required.2) How to ensure all the unmethylated C bases to be converted to T bases in the bisulfite treatment of DNA?Our experimental pipeline normally ensures the conversion rate of unmethylated C to T to meet the bioinformatics analysis requirement. Here we used standard DNA and H19 for quality control.3) How to classify the methylation level?We can classify the methylation level through the proportion of total C and total T sequenced in a certain region. For example, if we get 6C and 4T (which in the reference sequencing is C), the methylation level in this region is considered as 6/ (4+6)= 60%.4) Which factors will influence the result of bisulfite-seq?From the scientific view, uncertain factors of DNA methylation research are mainly from undiscovered area, such as methylation difference of individual cell from cell lines, time differences and dynamic changes of methylation during developmental process or pathological process; from the technical view, the length of sample DNA, the conversion rate of DNA during bisulfate treatment and sequencing depth may influence the result of bisulfate-seq.目前, 基因的甲基化研究主要结合亚硫酸氢钠处理和PCR技术，分为甲基化特异性PCR（Methylation specific PCR,MSP）和硫化测序PCR（Bisulfite sequencing PCR, BSP）。

30 Churchill Place ● Canary Wharf ● London E14 5EU ● United Kingdom Telephone +44 (0)20 3660 6000 Facsimile +44 (0)20 3660 555525 January 2016EMA/CHMP/ICH/310133/2008Committee for Human Medicinal ProductsICH guideline E14: the clinical evaluation of QT/QTc interval prolongation and proarrhythmic potential for non-antiarrhythmic drugs (R3) - questions and answersStep 5E14 Q&As (R3) Document HistoryICH guideline E14: the clinical evaluation of QT/QTc interval prolongation and proarrhythmic potential for non-antiarrhythmic drugs (R3) - questions and answersICH guideline E14 (R3) - questions and answersTable of contents1E lectrocardiograms methodology (4)2G ender (9)3P ositive control (10)4S tudy design (12)5U se of concentration response modeling of QTc data (13)6S pecial cases (16)7E lectrocardiograms monitoring in late stage clinical trials (17)ICH guideline E14 (R3) - questions and answers1Electrocardiograms methodologyICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 4/20ICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 5/20ICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 6/20ICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 7/20ICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 8/202GenderICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 9/203Positive controlICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 10/20ICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 11/204Study designICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 12/205Use of concentration response modeling of QTc dataICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 13/20ICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 14/20ICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 15/20ICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 16/20 6 Special cases6.1 March2014 The ICH E14 Guideline states that in certain cases a conventional thoroughQT study might not be feasible. In suchcases what other methods should beused for evaluation of QT/QTc andproarrhythmic potential? In certain cases the conventional “thorough QT/QTc” study design (a crossover study in healthy volunteers with short-term administration of the usual maximum dose and one higher dose with placebo and positive control) might need to be modified for a drug or active metabolite with a long half-life or delayed QT effect, or because of safety, tolerability or practical issues that preclude use in healthy subjects. In most cases alternative designs can be used that may affect power considerations, but do notcompromise study interpretation. For example, multiple doses can be studied in aparallel design trial or can use patients with the disease for which the drug is intendedrather than healthy volunteers.Where a placebo-controlled comparison using appropriate doses is not possible,alternative study d esigns should incorporate as many of the usual “thorough QT/QTc”design features as possible, and the quality and extent of the pre-clinical evaluation (ICHS7B Guideline) is particularly critical. Other useful supplementary data might includeintensive ECG data acquisition in early phase single or multiple ascending dose studies,utilisation of concentration-response analysis, and evaluation of exposures that aregreater than are anticipated with the intended marketed dose.A single dose of a positive control is generally sufficient, even if it precedes theinvestigational drug treatment. In the absence of a positive control, there is reluctanceto draw conclusions of lack of an effect; however, if the upper bound of the two-sided90% confidence interval around the estimated maximal effect on QTc is less than 10 ms,it is unlikely to have an actual mean effect as large as 20 ms.When a thorough QTc study of usual or modified design is not feasible, the intensity oflate phase ECG monitoring will be dependent upon the quality and extent of the non-clinical and clinical evaluation. In situations where it is not possible to study higherexposures than are anticipated with the intended marketed dose, more intensive ECGmonitoring might be necessary during Phase 3 trials. When the non-clinical and early7Electrocardiograms monitoring in late stage clinical trialsICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 17/20ICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 18/20ICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 19/20ICH guideline E14 (R3) - questions and answersEMA/CHMP/ICH/310133/2008 Page 20/20。

COMMISSION DECISIONof 28 May 2009amending Council Directive 76/769/EEC as regards restrictions on the marketing and use of organostannic compounds for the purpose of adapting its Annex I to technical progress(notified under document number C(2009) 4084)(Text with EEA relevance)(2009/425/EC)THE COMMISSION OF THE EUROPEAN COMMUNITIES,Having regard to the Treaty establishing the EuropeanCommunity,Having regard to Council Directive 76/769/EEC of 27 July 1976 on the approximation of the laws, regulations and admin ­istrative provisions of the Member States relating to the restrictions on the marketing and use of dangerous substancesand preparations ( 1 ), and in particular Article 2a thereof, Whereas:(1) Tri-substituted organostannic compounds werepreviously widely used in antifouling paints on ships. However, such paints were found to pose risks for aquatic organisms through endocrine disruptive effects. The use of organostannic compounds, also known as organotin compounds, in antifouling paints was therefore restricted in Directive 76/769/EEC, and in Regulation (EC) No 782/2003 of the European Parliament and of the Council of 14 April 2003 onthe prohibition of organotin compounds on ships ( 2 ).Furthermore, tri-substituted organostannic compounds may no longer be used as biocides under Directive 98/8/EC of the European Parliament and of the Council of 16 February 1998 concerning the placing of biocidalproducts on the market ( 3 ). However articles treated withsuch biocides may still be imported into the Community.(2) Di-substituted organostannic compounds, including inparticular dibutyltin compounds (DBT) and dioctyltin compounds (DOT), are widely used in consumer articles where they function either as a stabilizer or as a catalyst.(3) The use of organostannic compounds in consumerarticles has been found to pose a risk to human health, particularly for children. The specific risks to the health of children and adults from various consumer articleshave been identified in a risk assessment ( 4 ), and havebeen confirmed by the Commission’s ScientificCommittee on Health and Environmental Risks (SCHER) in its opinion of 30 November 2006 ( 5 ).(4) Although di-substituted and tri-substituted organostanniccompounds have the same adverse health effect, namelyimmunotoxicity via the thymus gland, and act in a cumulative way, the potency of the tri-substituted compounds (such as TBT and TPT) is greater than that of the di-substituted compounds (DOT and DBT). Furthermore, tri-substituted organostannic compounds being emitted from articles for either consumer or professional use could have adverse effects on the environment — in particular on aquatic organisms. More severe restrictions should therefore be imposed on articles containing tri-substituted organostannic compounds.(5) Certain DBT compounds (dibutyltin dichloride, CAS:683-18-1, and dibutyltin hydrogen borate, CAS:75113-37-0) will soon be classified in the framework of Council Directive 67/548/EEC ( 6 ) as toxic to repro ­duction, category 2, and it will subsequently be prohibited to sell the substances and mixtures containing them to consumers ( 7 ). More severe restrictions should therefore be imposed on articles containing DBT compounds, allowing for continued use for an additional period of time only where no suitable alternatives are available such as catalysts in RTV-1 and RTV-2 sealants, paints and coatings, or PVC stabilisers in certain products (for example coated fabrics, PVC profiles) to allow the development of suitable alter ­natives, or where the articles concerned are already regulated by other more specific legislation.(6) Exposure to DOT compounds is highest from certainspecific consumer articles such as printed textiles,gloves, footwear, wall and floor coverings, female hygiene products, nappies, and two-component silicone moulds.(7) Despite the availability of alternatives for most uses to berestricted, certain producers of articles containing DOT and DBT will need time to adapt, and an appropriate transition period should be therefore foreseen for theseapplications.Journal of the European Union L 138/11( 1 ) OJ L 262, 27.9.1976, p. 201.( 2 ) OJ L 115, 9.5.2003, p. 1. ( 3 ) OJ L 123, 24.4.1998, p. 1.( 4 ) Risk assessment studies on targeted consumer applications of certain organotin compounds. RPA study finalised in September 2005. http://ec.europa.eu/enterprise/chemicals/studies_en.htm( 5 ) http://ec.europa.eu/health/ph_risk/committees/04_scher/ scher_opinions_en.htm ( 6 ) OJ 196, 16.8.1967, p. 1.( 7 ) In accordance with entries 29, 30 and 31 of Directive 76/769/EEC.(8) Directive 76/769/EEC should therefore be amendedaccordingly.(9) This Decision is without prejudice to the Communitylegislation laying down minimum requirements for theprotection of workers, such as Council Directive89/391/EEC of 12 June 1989 on the introduction ofmeasures to encourage improvements in the safety andhealth of workers at work (1) and individual Directivesbased thereon, in particular Directive 2004/37/EC ofthe European Parliament and of the Council of29 April 2004 on the protection of workers from therisks related to exposure to carcinogens or mutagens atwork (Sixth individual Directive within the meaning ofArticle 16(1) of Council Directive 89/391/EEC) (codifiedversion) (2) and Council Directive 98/24/EC of 7 April1998 on the protection of the health and safety ofworkers from the risks related to chemical agents atwork (fourteenth individual Directive within themeaning of Article 16(1) of Directive 89/391/EEC) (3).(10) The measures provided for in this Decision are inaccordance with the opinion of the Committee on theadaptation to technical progress of the Directives for theelimination of technical barriers to trade in dangeroussubstances and preparations,HAS ADOPTED THIS DECISION:Article 1Annex I to Directive 76/769/EEC is amended in accordance with the Annex to this Decision.Article 2This Decision is addressed to the Member States.Done at Brussels, 28 May 2009.For the CommissionGünter VERHEUGENVice-PresidentL 138/12 Official Journal of the European Union 4.6.2009(1) OJ L 183, 29.6.1989, p. 1.(2) OJ L 158, 30.4.2004, p. 50; corrected by OJ L 229, 29.6.2004,p. 23.(3) OJ L 131, 5.5.1998, p. 11.ANNEXThe following paragraphs are added to Entry 21, ‘Organostannic compounds’, of Annex I to Directive 76/769/EEC:Journal of the European Union L 138/13。

UNIT 2.12SNP Genotyping Using the Sequenom MassARRAY iPLEX PlatformStacey Gabriel,1Liuda Ziaugra,1and Diana Tabbaa 11Broad Institute of MIT and Harvard,Cambridge,MassachusettsABSTRACTThe method for SNP genotyping described in this unit is based on the commercially available Sequenom MassARRAY platform.The assay consists of an initial locus-specific PCR reaction,followed by single base extension using mass-modified dideoxynucleotide terminators of an oligonucleotide primer which anneals immediately upstream of the polymorphic site of ing MALDI-TOF mass spectrometry,the distinct mass of the extended primer identifies the SNP allele.Curr.Protoc.Hum.Genet.60:2.12.1-2.12.18.C 2009by John Wiley &Sons,Inc.Keywords:genotyping r SNP r MassARRAY r high-throughput r PCRINTRODUCTIONMany high-throughput single-nucleotide polymorphism (SNP)genotyping technologies are currently available.Each offers a unique combination of scale,accuracy,throughput,and cost.However,the state of SNP genotyping is in flux,with no single technology or platform that can satisfy all users and study designs.The Sequenom MassARRAY plat-form has several attractive features for users desiring an accurate custom SNP genotyping assay (Gabriel et al.,2002)with modest multiplexing and minimal assay setup costs due to unmodified oligonucleotide primers.It utilizes a homogeneous reaction format with a single extension primer to generate allele-specific products with distinct masses,multi-plexed PCR reactions,a single termination mix and universal reaction conditions for all SNPs,small reagent volumes,and a throughput of >100,000genotypes/day/system.The method described in this unit is based on the commercially available Sequenom MassARRAY platform and reflects modifications made by the authors in establishing a high-throughput genotyping laboratory utilizing four MassARRAY systems.The assay (Tang et al.,1999)is based on primer extension and offers two levels of specificity.First,a locus-specific PCR reaction (see Basic Protocol 2)takes place,followed by a locus-specific primer extension reaction (iPLEX assay;see Basic Protocol 3)in which an oligonucleotide primer anneals immediately upstream of the polymorphic site being genotyped (see Basic Protocol 1for preparation of probe and genomic DNA).In the iPLEX assay,the primer and amplified target DNA are incubated with mass-modified dideoxynucleotide terminators (Fig.2.12.1).The primer extension is made according to the sequence of the variant site,and is a single complementary mass-modified base.Through the use of MALDI-TOF mass spectrometry,the mass of the extended primer is determined.The primer’s mass indicates the sequence and,therefore,the alleles present at the polymorphic site of interest.Sequenom supplies software (SpectroTYPER)that au-tomatically translates the mass of the observed primers into a genotype for each reaction.BASIC PROTOCOL 1PRE-PCR:DNA AND OLIGO POOL PREPARATIONThis protocol describes preparation of working DNA and oligonucleotide plates for genotyping on the Sequenom platform.Current Protocols in Human Genetics 2.12.1-2.12.18,January 2009Published online January 2009in Wiley Interscience ().DOI:10.1002/0471142905.hg0212s60Copyright C 2009John Wiley &Sons,Inc.Genotyping 2.12.1Supplement 60SNP Genotyping Using Sequenom MassARRAY iPLEX Platform2.12.2Supplement 60Current Protocols in Human Genetics 5500600065007000Mass 7500800085005000Figure 2.12.1The MassEXTEND iPLEX reaction.A schematic of the genotype reaction of a C-to-G SNP .Genotyping 2.12.3Current Protocols in Human Genetics Supplement 60DNAGenomic DNA,whole-genome amplification (WGA)DNA,or cDNA samples are dilutedto 2.5to 5ng/μl concentration in TE buffer at a concentration of 0.25×or less (the UVA 260/A 280ratio should be between 1.7and 2.0).The DNA is divided into aliquots (alsosee Basic Protocol 2,step 1)at 2μl/well into a 384-well PCR reaction plate from adeep-well PCR source plate (Marsh Biomedical).Packard or Tecan 8-tip robotics can beused for DNA dilution and transfer into the deep-well source plate.96-or 384-tip robotscan be used to transfer the 2-μl DNA aliquot into the wells of the 384-well PCR reactionplate.DNA needs to be amplified by PCR prior to performing an extension reaction ofthe SNP of interest.Keep DNA-containing plates at 4◦C.OligonucleotidesSequenom’s MassARRAY Designer software automatically designs PCR and extensionprimers (probes)for each SNP to be investigated (see Background Information).Alloligos for PCR and iPLEX reactions are ordered unmodified,with standard purification(e.g.,Integrated DNA Technologies).Forward and reverse PCR primers are ordered in96-well deep-well plates,mixed by the vendor at final,equimolar 240μM concentrations.Collapsed PCR primers undergo further dilution after the pooling to a working concen-tration of 1μM each.Probes (for iPLEX extension)are ordered unmixed in 96-welldeep-well plates at 250to 450μb technicians or automation robots perform PCRand extension oligo pooling according to the assay pool plexes and oligo plate maps.The PCR assay pool plexes consist of the multiplexed forward and reverse PCR oligonu-cleotide primers for each reaction present together in one multiplexed assay pool.TheSingle Base Extension pool plexes consist of the multiplexed oligonucleotide primers,which anneal adjacent to the polymorphic site for each reaction present together in themultiplexed assay pool.By pooling locus-specific primers,many individual loci of DNAwith their corresponding SNP sites may be analyzed in a one-well reaction.Due tothe inverse relationship between peak intensity and analyte mass,extension primers iniPLEX assays are adjusted by concentration to ensure that the extension primers are asequal in intensity as possible.The primers are sorted into three groups based on mass,with the highest mass group diluted to 15μM,the middle group to 10μM,and the lowmass group to 5μM.Extension oligo pools are spotted on SpectroCHIPs and run on de-tectors to verify uniformity of intensities of low/medium/high mass probes and accuracyof the manual pooling procedure (probes’masses must match expected spectra).Anyoligos of low-peak intensity are spiked in by adding volume to equalize the probe peakintensities.For example,if a given peak is about half the intensity of the other peaks,anequal volume is spiked in to double the peak intensity (see Fig.2.12.2and below).Storediluted working oligos at 4◦C and concentrated stocks at −20◦C.Adjusting Oligo Concentration within Each Assay Pool1.Divide the available volume of the highest mass oligo Bin by 3to get the volume forthe lowest mass Bin 1,and divide by 1.5to get the volume for the second Bin 2.2.Determine the final volume by multiplying the volume of Bin 3(highest mass oligos)by the initial oligo concentration and dividing by 15μM.3.Multiply the volumes obtained for each bin by the number of oligos in each bin andadd these values together.Subtract this total from the final volume to get the volumeof water (if any)to add.4.Add 5μl of the oligo pool mix to 95μl water.Mix well.5.Aliquot 20μl to several wells of a 384-well PCR plate (one pool per row).Definethe plate in the Sequenom Typer Plate Editor database as to which pools are in whichwells.Add resin,and run on the Mass Spectrometer as done for sample plates.SNP Genotyping Using SequenomMassARRAY iPLEX Platform2.12.4Supplement 60Current Protocols in Human Genetics A B bin 1=5μM:1×bin 2=10μM:2×bin 3=15μM:3×non-adjusted equimolar primer mix adjusted I n t e n s i t y I n t e n s i t y Mass Mass Figure 2.12.2Adjustment of extend oligo concentrations within each pool.(A )Spectra of ad-justed and non-adjusted oligos in an assay pool (figure from Sequenom’s iPLEX Gold Application Guide).The arrows indicate obvious peak intensity differences before and after primer concentra-tion adjustment.(B )Sorting of the oligos into three bins,with oligo masses increasing within each pool from left to right by column and then downwards by row within the plate array.The three bins within each separate pool are shown with varying color intensity for distinction.Bin 1,being the darkest,is pooled at the lowest concentration,bin 2is pooled at medium concentration,and bin 3,being the lightest,is pooled at the highest concentration.6.Examine the relative peak heights in Typer over several wells to determine if any extend oligos need to be spiked in amount (generally done for peaks that have less than half the intensity of other peaks).Add a corresponding amount of that particular extend oligo to the pool.For example,add an equal amount of the original amount to double the concentration of that oligo.BASICPROTOCOL 2AMPLIFICATION OF TARGET LOCI BY PCR This procedure describes the steps involved in amplifying a specific fragment of genomic DNA for the purpose of genotyping that fragment on the Sequenom platform.The goal of an optimal multiplex PCR reaction is to evenly amplify many individual loci of DNA with minimal nonspecific byproducts.Purified amplicons are then used as templates for the primer extension reaction (see Basic Protocol 3).This protocol describes setup of reaction plates,either when one primer pool is applied across 384DNA samples (Fig.2.12.3A),or when four primer pools are used for 96DNA samples in offset quadrants on 384-well PCR plates (Fig.2.12.3B).Due to the configuration of the Multimek robot describedGenotyping 2.12.5Current Protocols in Human Genetics Supplement60Figure 2.12.3Recommended configurations for setup of 384-well PCR plate.(A )Same assaypool for all wells,different DNA in the wells;(B )Same 96DNA samples arrayed in offset quadrantwells,four different assay pools.below,the “tight-quadrant”format requires specialized automation (or manual transfer)for transfer of primer pools to the 384plate.This protocol is intended for assaying at least one 384-well plate,and can be scaled upto encompass many plates.NOTE:As with all PCR methodology,exercise great care to ensure a lack of contam-ination of the preparations with unwanted DNA.A separate “pre-PCR”area should bereserved for setting up PCR (see APPENDIX 2D for further discussion of special considera-tions for PCR experiments).MaterialsDNA source plate:384-well deep-well PCR plate containing 2.5ng/μl DNA ofinterest (see Basic Protocol 1;store at 4◦C)100mM dNTPs (Applied Biosystems;store at −20◦C)25mM MgCl 2(QIAGEN;store at −20◦C)Ultrapure PCR-grade H 2O (Invitrogen)5U/μl HotStarTaq Plus DNA polymerase with 10×PCR buffer (QIAGEN;store at −20◦C)Forward and reverse primers:1μM each in multiplex pool (see Basic Protocol 1and Background Information;store at 4◦C)384-well PCR reaction plate (Eppendorf twin.tec)96-tip or 384-tip pre-PCR Tomtec or Hydra-type (Matrix Technologies)roboticworkstation (dedicated to DNA)SNP GenotypingUsing SequenomMassARRAY iPLEX Platform2.12.6Supplement 60Current Protocols in Human Genetics96-tip pre-PCR Multimek robotic pipettor with stacker (Beckman-Coulter)and disposable aerosol-barrier “Beckman-style”tips (LabSource)96-well ABGene Thermo-Fast skirted plates (AB-0800)1.5-ml microcentrifuge tubes or 15-or 50-ml conical polypropylene centrifuge tubes (e.g.,Falcon)MicroAmp adhesive plate sealers (Applied Biosystems)Pre-PCR tabletop centrifuge with microtiter plate carriers ABI Viper dual or Thermo Scientific Hybaid 384-well blocks,PC-controlled thermal cyclers Add DNA to reaction plates 1.Create program for robotic workstation (Tomtec or Hydra-type dedicated to DNA)to dispense 2μl of DNA sample per well of 384-well PCR plate,using either of the configurations shown in Figure 2.12.3:384DNA samples to 384-well PCR plate applying one pool per plate or 96DNA samples to 384-well PCR plate with offset quadrants,applying four pools.Dispense the DNA in the selected pattern Add PCR master mix to reaction plate 2.Create program on pre-PCR Multimek automated pipettor that allows addition of 4μl of master mix from a 96-well ABGene plate into each well of the 384-well reaction plate.The V-bottom ABGene source plate allows for lower source plate volumes.An example layout of a Multimek with plate stackers is shown in Figure 2.12.4.3.Calculate the volumes needed for the number of wells in the plate for each assay pool from Table 2.12.1,corresponding to the dispensed DNA pattern chosen from Figure 2.12.3(also see step 1)bine PCR master mix components in the amounts and in the order shown in Table 2.12.1in 1.5-ml microcentrifuge tubes or 15-to 50-ml conical polypropylene centrifuge tubes,depending on the number of plates and assays to be run.For the offset quadrants,make four mixes using four primer pools.Aliquot the appropriate volume per well into the 96-well ABGene source plate.IMPORTANT NOTE :If running more than twelve 384-well plates,it is necessary to prepare two plates of master mix,because the ABGene plates should not be used to hold >220μl per well.Wear gloves when working in pre-PCR area.5.Calibrate the Multimek pipettor according to the manufacturer’s instructions.Make sure that the wash bucket is full of water and the waste bucket is empty before beginning.Put on aerosol-resistant tips.6.Put 384-well PCR plates containing the source DNA (see step 1)in the plate carriers and place in the left-most stacker.7.Place PCR mix,in an ABGene plate,in Position 1on the robot deck.8.Open the Start menu,select Programs,and then open the Stacknet Software.Press Start when the Stacknet window opens up.9.From the Stacknet window,select Program,and then select the Computer radio button.10.In the Computer menu,press Load,then select the program that was created to add 4μl of master mix (see step 2).Press Start.The program will “error out”if the robot cannot pick up any more plates.The total reaction volume per well will be 6μl:4μl of master mix and 2μl of 2.5ng/μl source DNA.Genotyping 2.12.7Current Protocols in Human Genetics Supplement 60Table 2.12.1PCR Master Mix aH 2OPCR buffer (10×)MgCl 2dNTP mix Primer mix (1μM)HotStarTaq Plus Total Amount per well of 96-well plate 1well2.2220.8250.4290.1320.660.132b 4.4c 1plate 1194.55443.52230.6370.96354.8270.962365.4422μl a Reagent volumes in microliters.b For iPLEX Gold >24plex reaction,increase Taq to 0.220μl per well.c Only 4μl is added to each well.right-handstackerleft-hand stacker tip-rack change tip-washwater bathplate position 1plateposition 2plate running track Figure 2.12.496-tip head Beckman Multimek with stacker deck layout.This configuration isused throughout the protocol.11.Empty the waste bucket and place the wash tube into a new water bottle at the endof the run.Remove the reaction plates from the stacker,and carefully remove themfrom the carriers.Seal plates carefully with MicroAmp sealers.Dispose of the usedABGene plate.V ortex the plates on a plate vortexer.12.Centrifuge the plates 1min at 425×g ,room temperature,in a pre-PCR tabletopcentrifuge with microtiter plate carriers,to bring the solutions to the bottom of thewells.Perform thermal cycling (PCR reaction)13.Place the plates in the ABI or Hybaid 384-well block.Set up lid mode as Constant at 100◦C.Enter 6μl volume.Perform PCR with the following cycling program (runs∼2.5hr):1cycle:5min 94◦C (initial denaturation)45cycles:20sec 94◦C (denaturation)30sec 56◦C (annealing)1min 72◦C (extension)1cycle:3min 72◦C (final extension)Final step:indefinite 4◦C (hold).14.Centrifuge the plates as in step 12to bring the solutions to the bottoms of the wells,then store at 4◦C until ready for SAP cleanup procedure (Support Protocol 1).SNP Genotyping Using SequenomMassARRAY iPLEX Platform2.12.8Supplement 60Current Protocols in Human GeneticsSUPPORTPROTOCOL 1POST-PCR:SAP REACTION CLEANUP Shrimp alkaline phosphatase (SAP)dephosphorylates unincorporated dNTPs by cleaving the phosphate groups from the 5 termini.Treatment with SAP is performed in order to remove remaining,nonincorporated dNTPs from amplification products.This procedure is performed on a post-PCR 96-tip SpectroPREP Multimek robot.The Multimek dispenses 2μl of a SAP cocktail into each individual well of the 384-well post-PCR reaction plate.After SAP cocktail is added,the plate is removed from the robot and centrifuged.Treated plates are placed in a 37◦C incubator for 50min,and right after this are placed in an 85◦C incubator for an additional 20min to inactivate SAP enzyme.Alternatively,incubation can take place on a 384-well block thermal cycler.Materials 1×SAP buffer (Sequenom)1.7U/μl shrimp alkaline phosphatase (SAP;Sequenom;store at 0◦C)PCR products in 384-well PCR plates (see Basic Protocol 2)1.5-ml microcentrifuge tubes or 15-or 50-ml conical polypropylene centrifuge tubes (e.g.,Falcon)96-well ABGene plates (AB-0800)96-tip post-PCR Multimek robotic pipettor with stacker (Beckman-Coulter)MicroAmp adhesive plate sealers (Applied Biosystems)Post-PCR tabletop centrifuge with microtiter plate carriers 37◦and 85◦C Precision incubators (VWR)or ABI or Hybaid thermal cycler with 384-well blocks Set up SAP reaction bine SAP reaction components in the amounts and in the order shown in Table 2.12.2in 1.5-ml microcentrifuge tubes or in 15-to 50-ml conical polypropylene centrifuge tubes,depending on the number of plates and assays to be run.2.Based on the number of plates to be run,aliquot the correct amount of SAP reaction cocktail per well of a 96-well ABGene plate.3.Place the 384-well reaction plates onto carriers and load them onto the stackers in the post-PCR Multimek SpectroPREP robot.Place the 96-well ABGene plate with the SAP cocktail on to the Multimek in Position 1.4.Select the SAP program from the StakNet Control Program to add 2μl of SAP cocktail to each well.Once the 2μl of cocktail is dispensed,the robot then mixes the 6μl of post-PCR product that was already in each of the wells with the 2μl of added SAP cocktail,creating a homogeneous mixture.5.Remove the reaction plates from the stacker and carefully remove them from the car-riers.Seal the plates carefully with MicroAmp sealers.Dispose of the used ABGene plate.V ortex the plates on a plate vortexer.Table 2.12.2SAP Reaction Cocktail a Water 10×SAP buffer SAP (1.7U/μl)Total amount of mix 1well 1.530.170.321plate 1107.72123.08217.21448a Reagent volumes in microliters.Genotyping 2.12.9Current Protocols in Human Genetics Supplement 606.Centrifuge the plates 1min at 425×g ,room temperature,in a post-PCR tabletopcentrifuge with microtiter plate carriers,to bring solutions to the bottom of the wells.Incubate SAP reactionsUsing incubators7a.Incubate the plates in a 37◦C Precision incubator for 50min,then immediately transfer to an 85◦C incubator and incubate for 20min.IMPORTANT NOTE :Do not stack plates in the incubators;spread them in one layeronly.8a.Remove the plates from the 85◦C incubator and cool to room temperature.Centrifugeas in step 6,then store at 4◦C until ready to process for primer extension procedure(see Basic Protocol 3).Using thermal cycler7b.Place the plates in the ABI or Hybaid 384-well block.Execute the followingprogram:1cycle:40min 37◦C1cycle:10min 85◦CFinal step:indefinite 4◦C.8b.Centrifuge as in step 6,then store at 4◦C until ready to process for primer extensionprocedure (see Basic Protocol 3).BASIC PROTOCOL 3PRIMER EXTENSIONThe primer extension or iPLEX reaction is a universal method for detecting single-base polymorphisms or small insertion/deletion polymorphisms in amplified DNA.AfterPCR cleanup (Support Protocol 1),a primer extension reaction cocktail (containingextend primer,buffer,enzyme,and mass-modified ddNTPs)is added to the amplificationproducts.During the iPLEX reaction,the primer is extended by one mass-modifiednucleotide depending on the allele and the design of the assay.This protocol describes the reaction plate setup when either one primer pool is appliedacross 384DNA samples or four primer pools are used for 96DNA samples in offsetquadrants on 384-well PCR plates (see Fig.2.12.3A and B).This protocol uses aMultimek robot.The reaction cocktail is easy to scale up for any number of PCRplates.MaterialsUltrapure PCR-grade H 2O (Invitrogen)iPLEX enzyme (Sequenom)10×iPLEX buffer (Sequenom)iPLEX Extension Mix (Sequenom)Extend primers:from 5to 10μM each in multiplex poolPCR products in 384-well PCR plates,cleaned up by SAP reaction (see SupportProtocol 1)1.5-ml microcentrifuge tubes or 15-or 50-ml conical polypropylene centrifugetubes (e.g.,Falcon)96-well ABGene plates96-tip post-PCR Multimek SpectroPREP (Sequenom)with stackerMicroAmp adhesive plate sealers (Applied Biosystems)Post-PCR tabletop centrifuge with microtiter plate carriersABI or Hybaid Thermal cycler with 384-well blocksSNP GenotypingUsing SequenomMassARRAY iPLEX Platform2.12.10Supplement 60Current Protocols in Human Genetics Table 2.12.3Primer Extend Master Mix with Sequenom T ermination MixesH 2OiPLEX buffer (10×)iPLEX extension mix Probe mix in 3bins (5μM to 15μM)iPLEX enzyme Total Amount per well of 96-well plate 1well0.49260.2220.2 1.04440.04121plate 346.79156.29140.8735.2628.86140813μl a Reagent volumes in microliters.Set up primer extension reaction 1.Calculate the amount of cocktail needed based on the number of wells,and combine primer extension reaction components in the amounts and in the order shown in Table 2.12.3in 1.5-ml microcentrifuge tubes or in 15-to 50-ml conical polypropylene centrifuge tubes,depending on the number of plates and assays to be run.Add primer extension cocktails to reaction plates 2.Aliquot cocktail into 96-well ABGene plate in the configuration corresponding to the PCR plate layout (see Basic Protocol 2and Fig.2.12.3).Make four mixes for the offset plate pattern.3.Place the 384-well reaction plates (from Support Protocol 1)on to carriers,and load them on to the stackers.4.Place the ABGene plate with the primer extension cocktail (from step 2)on to the Multimek in Position 2.5.Select the iPLEX Cocktail Addition program from the StakNet Control Program and run the program to add 2μl of primer extension mix to each well of the reaction plate.6.Remove the reaction plates from the stacker and carefully remove them from the carriers.Seal plates carefully with MicroAmp sealers.Dispose of the used ABGene plate.V ortex the plates on a plate vortexer.7.Centrifuge the plates 1min at 425×g ,room temperature,in the post-PCR cen-trifuge with microtiter plate carriers to bring the solutions to the bottom of the wells.Perform thermal cycling (primer extension reaction)8.Place plates in the ABI or Hybaid 384-well block.Perform primer extension with the following cycling program:1cycle:30sec 94◦C (initial denaturation)40cycles:5sec 94◦C (denaturation)5cycles:5sec 52◦C (annealing)5sec 80◦C (extension)1cycle:3min 72◦C (final extension)Final step:indefinitely 4◦C (hold).Note that the 5cycles sit within the 40cycles.9.Clean up the primer extension productions (see Support Protocol 2).Genotyping2.12.11SUPPORT PROTOCOL 2PRIMER EXTENSION REACTION RESIN CLEANUPThis cleanup step is important to optimize mass spectrometry analysis of the extended reaction products.SpectroCLEAN (Sequenom)is a cationic resin pretreated with acid reagents.A slurry of the resin is added directly to primer extension reaction products to remove salts such as Na +,K +,and Mg 2+ions.If not removed,these ions can result in high background noise in the mass spectra.This procedure takes ∼1.5hr for ten plates.MaterialsUltrapure PCR-grade H 2O (Invitrogen)SpectroCLEAN resin (Sequenom;store at room temperature)Post–primer extension products in 384-well plates (see Basic Protocol 3),kept at 4◦C Clean plastic bottle with capTurbulator nonmagnetic mixing device (ACME Automation),firmly positioned on the Multimek deck (e.g.,Position 1)96-tip post-PCR Multimek SpectroPREP (Sequenom)with stacker Disposable “Beckman-style”tips for MultimekPost-PCR tabletop centrifuge with microtiter plate carriers TiterTop plate sealers (LabSource)Plate rotator (VWR)Make resin slurry1.Add 80ml of ultrapure water to a clean plastic bottle reserved for resin.Transfer resin contents from two whole containers (28g each)of SpectroCLEAN resin into the bottle with the water.2.Cap the bottle and swirl it gently a few times to disperse all resin particles as evenly as possible in the suspension.Wait a little bit and swirl again,making sure that all entrapped air is gone.3.Swirl again and pour enough of the resin suspension into a Turbulator to just cover the bottom.Apply pressurized air,letting it go through the resin slurry.Slowly add more of the resin,mixing thoroughly.Remove air bubbles with a clean pipet tip.The Turbulator is a nonmagnetic mixing device.Operating via a vacuum diaphragm pulse technique,it pulses ∼4times a second with a fully adjustable amplitude,thereby creating a mild to major effect with 96little geysers that can protrude above the surface of the reservoir liquid.The system includes a high-quality vacuum pump,replaceable diaphragm,and tubing.Make sure Turbulator is firmly positioned on Multimek deck,for example,at Position 1(Fig.2.12.4).Calibrate SpectroPREP Multimek and load/unload tips4.Calibrate the instrument by opening the Communication Engine icon on the computer desktop designated for Multimek use and selecting Calibrate under the Multimek column.5.Load or unload tips by double-clicking on the Method Editor icon and selecting tip load or unload.Make sure that the tip rack is placed towards the back to load tips or forward to unload tips.Click OK.Go to Script and select RUN.Add resin6.Begin an application by going to Start and selecting the StackNet Stacker Network Control option under Programs.7.Select Start on the StackNet Plate Process Network Control screen.Select Program followed by Computer,then Load.SNP Genotyping Using Sequenom MassARRAY iPLEX Platform2.12.128.Centrifuge post-primer extension plates1min at425×g,room temperature,inpost-PCR tabletop centrifuge with microtiter plate carriers to bring solutions to the bottom of the wells.9.Carefully unseal plates.Place the plates on metal carriers.Stack the plates in theright-hand stacker and hit the START button in the application program window.The Multimek will aspirate16μl of resin suspension(containing7mg resin)and add this volume to each well of the384-well reaction plates in a quadrant pattern.Make sure that the water pump is running well and that enough water is present in the tip wash station.10.After resin transfer,take the plates off the stacker and seal with TiterTop seal.Inspect plates after resin addition to be sure that all wells have gotten the same amount of resin.Rotate plates11.Place the plates on a plate rotator,balanced in pairs.Rotate for30min.12.After the rotation is done,turn off the rotator and remove the plates.Centrifuge theplates3min at425×g,room temperature,in post-PCR tabletop centrifuge.Centrifuged plates are ready to be spotted on SpectroCHIPs(see Basic Protocol4).The resin remains at the bottom of the well.The purified product will be removed from the supernatant by the SpectroPoint.They can be stored at4◦C for later spotting and detection (stable up to1month).Rotate the plates again after cold storage.BASIC PROTOCOL4SPOTTING PRIMER EXTENSION PRODUCTS ON SpectroCHIPsThis protocol describes the arraying of the extended/desalted analyte products from 384-well microtiter plates on384-sample SpectroCHIPs.In order to incorporate oligonu-cleotides with the appropriate matrix for MALDI-TOF(3-hydroxypicolinic acid),a small volume(∼25nl)is arrayed onto existing matrix spots on the silica chip.This process uses the capillary action of slotted pins and contact dispensing for nanovolumes. Materials100%and50%ethanolPlates from primer extension,cleaned up with resin and centrifuged(see Support Protocol2)3-point calibrant(Sequenom)SpectroPOINT nanoliter sample dispensing instrument with appropriate software (Sequenom)SpectroCHIPs(Sequenom)Adhesive plate sealers(TiterTop from LabSource)1.Open the Spectropoint software on the PC.2.Precondition the main head and single pin with100%ethanol by clicking the Pre-condition tab.Select30min.Empty the trough by clicking the appropriate button and dumping100%ethanol in the trough.3.Empty and refill the sonicator with50%ethanol from thefill tank.Click on thebutton within the Precondition tab tofill the trough.4.Wash the main head ten times by selecting CLEAN MAIN HEAD while10cyclesare selected.5.Spin down resined plates(see Support Protocol2)and load onto microtiter stage twoat a time.。