TAKARA 高保真酶E说明书

产 品 说 明 书◆高保真PCR扩增试剂盒◆目录号1521◆使用手册◆实验室使用，仅用于体外高保真PCR扩增试剂盒目录号：1521目录编号包装单位152101 100次152102 500次产品组成、储存：组成100次500次Pfu（5 U/μl）50μl250μl10×Pfu Buffer（M g2+ Plus） 1 ml 3mldNT P（10mM each）100μl0.5 mlPCR 灭菌水 5 ml 25 ml储存：-20 ℃产品介绍：本试剂盒提供DNA高保真扩增所需要的基本试剂。

DNA扩增时，用户需要自行准备模板和扩增所需要的引物。

高温嗜热Pfu DNA聚合酶主要用于对保真度要求非常高的DNA扩增，这些扩增要求扩增的产物中不能出现突变等错误。

Taq DNA聚合酶的扩增性能很强，但是用Taq扩增的PCR产物中有难以避免的随机突变。

Pfu DNA聚合酶除了5′-3′的聚合特性外，还有3′-5′端外切酶活性, 具有校正DNA扩增过程中突变。

Pfu DNA的长片段扩增的能力不及Taq DNA聚合酶，客户如果用于长链PCR扩增，可选用本公司的Long Taq或者Taq Plus DNA 聚合酶。

举例说明：按下列组份配制PCR反应液Pfu（5 U/μl）0.5 μl10×Pfu PCR Buffer（Mg2+ Plus） 5 μldNTP Mixture（10 mM each） 1 μl模板DNA（质粒5-20ng，基因组100-500 ng ）？μl引物1（20 μM） 1 μl引物2（20 μM） 1 μl灭菌蒸馏水up to 50 μl混匀后，按下表的条件立即进行PCR循环次数反应阶段温度时间1 变性92-94°C2 分钟20-35 变性退火延伸92-94°C55°C72°C30秒45秒2分钟/kb1 延伸72°C 7分钟注意事项：1.Pfu扩增效率通常比Taq酶差，这是由于Pfu具有3′-5′的外切酶活性（高保真性）所引起的，不是由于Pfu酶的质量不稳定所致。

TaKaRa Code：D102ApMD®19-T Vector宝生物工程(大连)有限公司目录内 容 Page●制品说明 1●制品内容 1●保 存 1●纯 度 1●用 途 1●pMD®19-T Vector的结构 1 ●实验操作 2■Control DNA片段的克隆实验2■一般DNA片段的克隆实验2●相关说明 3 ●使用注意 4 ●Q&A4●制品说明pMD ®19-T Vector是一种高效克隆PCR产物（TA Cloning）的专用载体。

本载体由pUC19载体改建而成，在pUC19载体的多克隆位点处的Xba I和Sal I识别位点之间插入了Eco R V识别位点，用Eco R V进行酶切反应后，再在两侧的3＇端添加“T”而成。

因大部分耐热性DNA聚合酶进行PCR反应时都有在PCR 产物的3＇末端添加一个“A”的特性，所以使用本制品可以大大提高PCR产物的连接、克隆效率。

由于本载体以pUC19载体为基础构建而成，所以它具有同pUC19载体相同的功能。

此外，本制品中的高效连接液Solution I 可以在短时间内（约30分钟）完成连接反应，其连接液可以直接用于细菌转化，大大方便了实验操作。

本制品中的Control Insert（500 bp）还可以用于Control 反应。

本载体与pMD ®18-T Vector 相比，本制品的 β-半乳糖苷酶的表达活性更高，菌落显示蓝色的时间缩短，菌落显示的蓝色更深。

因此，克隆后更容易进行克隆体的蓝白筛选。

●制品内容pMD ®19-T Vector（50 ng/μl） 20 μl×1支 Control Insert（50 ng/μl）10 μl×1支 Solution I*75 μl×2支* 使用时请于冰中融解。

●保存： -20℃●纯度■ Control Insert 经克隆后的白色菌落中，有90%以上含有Insert DNA 片段。

JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/biotech_osaka@toyobo.jp1 F0934KKOD -Plus-KOD-201 200 U 200 reactionsStore at -20°C Contents[1]Introduction[2]Components[3]Quality testing[4]Primer design[5]Cloning of PCR products[6]Protocol1. Standard reaction setup2. Cycling conditions[7]Examples[8]Troubleshooting[9] References[10] Related productsC AUSIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe general laboratory precaution and utilize safety while using this kit.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/biotech_osaka@toyobo.jp 1[ 1 ] Introduction[ 2 ] Components [ 3 ] Quality Testing DescriptionKOD -Plus- is based on DNA polymerase from the hyperthermophilic Archaeon Thermococcus kodakaraensis KOD11) 2). KOD -Plus- exhibits excellent high PCR fidelity and efficiency. The enzyme solution of KOD -Plus- contains two types of anti-KOD DNA polymerase antibodies that inhibit polymerase and 3’J5’ exonuclease activity, thus allowing for Hot Start PCR3). KOD -Plus- generates blunt-end PCR products, due to 3’J5’ exonuclease (proof-reading) activity.Features-Hot Start technology, using anti-KOD DNA polymerase antibodies, results in highly efficient amplification (see Example 1).-KOD -Plus- enables the following amplifications (maximum): 21 kb from lambda phage DNA, 12 kb from human genomic DNA, and 7 kb from cDNA.-KOD DNA polymerase has strong 3’J5’ exonuclease (proof-reading) activity. The PCR error ratio of KOD -Plus- is approx. 80 times less than Taq DNA polymerase.Table. 1 PCR fidelity comparison of each PCR enzyme.*PCR fidelity was based on the mutation frequency of PCR products using a positive-selection base assay with the rpsL gene 4).This reagent includes the following components for 200 reactions:KOD -Plus- (1.0 U/μl) * 200 μl × 110× Buffer for KOD -Plus- 1.0 ml × 125 mM MgSO4 1.0 ml × 12 mM dNTPs 1.0 ml × 1*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize polymerase and 3’J5’ exonuclease activity.Quality check can be performed by amplifying the β-globin gene (3.6 Kb) and p53 gene (4.0 Kb).JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/biotech_osaka@toyobo.jp 2[ 4 ] Primer Design[ 5 ] Cloning ofPCR products [ 6 ] Protocol -Primers should be 22-34 bases with a melting temperature (Tm) over 60°C. For amplification of a long target, 25-34 bases with high Tm values (≥ 65°C) are recommended. PCR primers should be designed according to the general guidelines.-KOD-Plus- generates blunt-end PCR products, due to 3’J5’ exonuclease (proof- reading) activity. Therefore, the product can be cloned according to a blunt-end cloning method.-PCR products of KOD-Plus- should be purified prior to restriction enzyme treatments. The 3’J5’ exonuclease activity of KOD DNA polymerase remains after the PCR cycles.1. Standard reaction setupThe following procedure is designed for use with the components provided in this kit. Before preparing mixture, all components should be completely thawed, except for the enzyme solution.* Do not use dNTPs from other kits or companies.Notes:-For PCR reactions, thin-wall tubes are recommended. A total reaction volume of 50 μl is also recommended.-The addition of DMSO (final conc. 2-5%) might be effective for amplification of GC-rich targets. Decreased PCR fidelity has been confirmed to not take place with DMSO.-Contaminated RNA (used for cDNA) or genomic DNA inhibits the PCR reaction by chelating Mg2+. PCR should be performed using template DNA containing <100 ng RNA component.Component Volume FinalConcentration 10x Buffer for KOD -Plus- 5 μl 1x2mM dNTPs* 5 μl 0.2 mM each25mM MgSO4 2μl 1.0mM10pmol/μl Primer #1 1.5 μl 0.3 μM10pmol/μl Primer #2 1.5 μl 0.3 μMTemplate DNA X μlGenomic DNA 10-200 ng/50 μlPlasmid DNA 1-50 ng/50 μlcDNA ≤ 100 ng （RNA equiv.)/50 μl PCR grade water Y μlKOD-Plus- (1.0 U/μl) 1μl 1.0 U / 50 μlTotal reaction volume 50 μlJAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/biotech_osaka@toyobo.jp32. Cycling conditionsThe following cycling steps are recommended.Note : If the Tm value of the primer is under 73 °C, the 3-step cycle is recommended.*Tm value of the primer minus 5°C-10°CNotes:-Extension time should be set to 1min per 1 kb of target length.< 2-step cycle >Pre-denaturation: 94 °C , 2 min. Denaturation: 94 °C, 15 sec. Extension:68 °C, 1 min./kb< 3-step cycle >Pre-denaturation: 94 °C, 2 min. Denaturation: 94 °C, 15 sec.Annealing: Tm-[5-10]oC*, 30 sec.Extension:68 °C, 1 min./kb25-35 cyclesJAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/biotech_osaka@toyobo.jp4[ 7 ] ExamplesExample 1．Effect of Hot Start PCR on the generation of primer dimers.Example 2. Effect of addition of DMSO for GC-rich targets.M: 1kb Template: Human genomic DNA Target: TGF-βgene (GC%=70) 2kb Ladder Markers 1: KOD -Plus-, 0% DMSO 2: KOD -Plus-, 2% DMSO 3: KOD -Plus-, 5% DMSOM 1 2 3M 123456MJAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/biotech_osaka@toyobo.jp5[ 8 ] Troubleshooting[ 9 ] References1)Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M, and Imanaka T., Appl Environ Microbiol., 63: 4504-10 (1997)2)Hashimoto H, Nishioka M, Fujiwara S, Takagi M, Imanaka T, Inoue T and Kai Y , J Mol Biol ., 306: 469-77 (2001)3)Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem ., 126: 762-8 (1999)4)Fujii S, Akiyama M, Aoki K, Sugaya Y , Higuchi K, Hiraoka M, Miki Y , Saitoh N, Yoshiyama K, Ihara K, Seki M, Ohtsubo E and Maki H, J. Mol. Bio l., 289: 835-850 (1999)SymptomCauseSolutionLower annealing temperature increments to a maximum of Tm-10°C.Cycling conditions are not suitable.Increase the number of cycles by 2-5 cycles. Mg concentration is low Increase the Mg concentration to 1.2-2 mM. High GC content of target sequenceAdd DMSO 2-5%. <See Example 2>Check the quality of primers. Primer is not good.Redesign primers.Check the quality of template DNA. RNA inhibits amplification.No PCR product/low yield Quality and/or quantity of template DNA is not sufficient.Increase the amount of template DNA.Decrease the number of cycles by 2-5 cycles. Cycling condition is not suitable.Use step-down cycling.Check the quality of primers. Primer is not good.Redesign primers.Too much template DNA Reduce the amount of template DNA. Too much Mg Reduce MgSO 4 to 0.8 mM.Smearing/extra bandToo much enzymeReduce enzyme to 0.5-0.8 U/50 μl.Poor TA cloning efficiency PCR products have blunt- ends.Clone the PCR products according to general blunt- end cloning guidelines.JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/biotech_osaka@toyobo.jp6[ 10 ] Related productsProduct namePackage Code No. TArget Clone -Plus- 10 reactions TAK-201 10x A-attachment mix 25 reactions TAK-301 Ligation high Ver.2750 μl(100 reactions)LGK-201TArget Clone -Plus- is a high efficient TA cloning kit. The kit can be applied to the TA cloning of blunt-end PCR products amplified using KOD -Plus- [Code No. KOD-201] or KOD FX [Code No. KFX-101]. The kit contains pTA2 Vector, 2x Ligation Buffer, T4 DNA Ligase and 10x A-attachment Mix.10 x A-attachment mix is a reagent comprising anti-KOD DNA polymerase antibody specific to KOD 3’J 5’ exonuclease activity (proof-reading activity), as well as Taq DNA polymerase, which exhibits terminal transferase activity. PCR products from KOD -Plus- [Code No. KOD-201] and KOD FX [Code No. KFX-101] possess blunt ends due to 3’J 5’ exonuclease activity of the KOD DNA polymerase. The 10 x A-attachment mix allows for PCR products to acquire overhanging dA at the 3’-ends. Products with 3’-dA overhangs can be directly cloned into arbitrary T-vectors using ligation reagents, such as Ligation high Ver.2 [Code No. LGK-201].Fig. Principle of the 10 x A-attachment mixAnti-KOD DNA polymeraseAJAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/biotech_osaka@toyobo.jp 7NOTICE TO PURCHASER: LIMITED LICENSEUse of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to US Patent No. 4,889,818.The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim (such as the patented 5’ Nuclease Process claims in US Patents Nos. 5,210,015 and 5,487,972), no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.。

Protocol：　・Reaction mixture preparation: The reaction mixture should be prepared on ice, and all necessary reagents should be stored on ice during reaction assembly. Preparing the reaction on ice decreases non-specific amplification due to primer misannealing. Add the reagents to the reaction tube in the following order: Sterilized distilled water, 5× e2TAK Buffer, dNTP Mixture, e2TAK DNA Polymerase, Template DNA, Primer 1, Primer 2. Mix the final reaction mixture by pipetting. TaKaRa recommends starting the reaction as soon as possible after reaction assembly. 　・General composition of PCR reaction mixture (50μl) e2TAK DNA Polymerase 0.5μl 5×e2TAK Buffer 10μl dNTP Mixture (2.5 mM each) 4μl Template DNA < 100 ng Primer 1 0.2～0.3μM (final conc.) Primer 2 0.2～0.3μM (final conc.) Sterilized distilled water up to 50μl *Recommended template amount Human genomic DNA 5 ng ～100 ng（< 100 ng） E.coli genomic DNA 100 pg ～100 ng λDNA 10 pg ～10 ng Plasmid DNA 100 pg ～10 ng　･PCR condition (example) Important Note:　TaKaRa strongly recommends use of short (5 - 15 sec.) annealing times in e2TAK PCR amplifications. Longer annealing times may increase the likelihood of product smears upon gel electrophoresis. 98℃　10 sec.30 cycles72℃　1 min./kb or98℃　10 sec.68℃　1 min./kb30 cycles　Note:　This product is intended to be used for research purpose only. They are not to be used for drug or diagnostic purposes, nor are they intended for human use. They shall not to be used products as food, cosmetics, or utensils, etc.　Takara products may not be resold or transfered, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC.　If you require licenses for other use, please call at +81 77 543 7247 or contact from our website at .Produced by TAKARA BIOTECHNOLOGY (DALIAN) CO.,LTD.200805DaDescription： e2TAK is a versatile DNA Polymerase optimized for PCR amplification of DNA. e2TAK has higher amplification efficiency than T aq DNA polymerase, and contains a 3' - 5' exonuclease activity. It is suitable for a variety of standard PCR applications.Components： e2TAK DNA Polymerase 20μl 5×PCR T Buffer (Mg 2+plus) 400μl dNTP Mixture (2.5 mM each) 180μl Storage buffer： 50 mM Tris-HCl (pH 8.2 at 4℃) 100 mM　NaCl 0.1 mM EDTA 1 mM DTT 50% GlycerolPurity： Nicking, endonuclease, and exonuclease activities were not detected using supercoiled pBR322 DNA,λDNA, or λ-Hin d III digest DNA as substrates.Application：For DNA amplification via the Polymerase Chain Reaction(PCR)PCR products： The majority of the PCR products obtained using e2TAK DNA Polymerase will possess blunt ends, Thus, e2TAK products may be cloned directly into blunt-ended vectors. (If necessary, phosphorylate the PCR products before cloning.)PCR test： Good performance of DNA amplification by PCR was confirmed by using λDNA as the template (amplified fragment：10 kbp).Good performance of DNA amplification of a single copy gene by PCR was also confirmed by using human genome DNA as the template (amplified fragment：approx. 4 kbp).Supplied 5X e2TAK Buffer (Mg 2+ plus)Size : 400 μl / vial Mg 2+ concentration (5X) : 5 mM Storage : － 20 ℃Supplied dNTP MixtureMixture of dNTP, ready for use in PCR reactions without dilution.Size : 180μl / vialConsentration : 2.5 mM of each dNTP pH : pH 7 ~ 9Form : Dissolived in water (sodium salts)Purity : ≧98 % for each dNTP Storage : － 20 ℃e2TAK DNA Polymerase(Sample)Code No. RF001Q Size : 40 react.Supplied Reagents :5×e2TAK Buffer (Mg 2+ plus)dNTP Mixture (2.5 mM each)Lot No.Volume : μlExpiry Date :Shipping at -20℃Stored at -20℃。

PrimeSTAR ® HSDNA Polymerase with GC Buffer使 用 说 明 书TaKaRa Code：DR044A●包装量： 250 U●制品说明PrimeSTAR ® HS DNA Polymerase with GC Buffer适用于高保真扩增具有复杂二级结构的DNA模板（GC rich、重复序列等）。

PrimeSTAR ® HS DNA Polymerase具有极强的3’→5’Exonuclease活性，显示出超群的校正功能，同时还具有优于Taq DNA Polymerase的高扩增效率。

本制品对cDNA克隆等要求高保真的PCR反应能够发挥强大威力,并能对高GC含量模板进行高效扩增。

使用本制品扩增得到的PCR产物几乎都为平滑末端，经磷酸化后可直接克隆于具有平滑末端的载体中。

● 制品内容* Mg 2+浓度为2 mM。

●保 存 -20℃。

●活性定义用活性化的大马哈鱼精子DNA 作为模板/引物，在74℃，30分钟内，摄入10 nmol 的全核苷酸为酸性不溶物的活性定义为1个活性单位（U）。

●纯 度1）10 U 的本酶和0.6 μg 的λDNA-Hin d III 在74℃下反应1小时，DNA 的电泳谱带不发生变化。

2）10 U 的本酶和0.6 μg 的SupercoiledpBR322 DNA 在74℃下反应1小时，DNA 的电泳谱带不发生变化。

●用 途PCR 法高保真扩增DNA 片段。

●PCR 反应性能1） 以λDNA 为模板，可以很好地扩增8、10、12、15 kbp 的DNA 片段。

2） 以人基因组DNA 为模板，可以很好地扩增APO E 基因520 bp（GC 含量74.8%）的DNA 片段。

●反应条件 1. 按下列组份配制PCR 反应液。

2×PrimeSTAR ® GC BufferdNTP Mixture（2.5 mM each）Primer 1 Primer 2Template*PrimeSTAR ® HS DNA Polymerase（2.5 U/μl） dH 2O*【50 μl PCR 反应体系中模板DNA 推荐使用量】人基因组DNA 大肠杆菌基因组DNA λDNA 质粒DNA cDNA Library25 μl4 μl 0.2-0.3 μM （final conc.） 0.2-0.3 μM（final conc.）<200 ng0.5 μlPrimeSTAR ® HS DNA Polymerase（2.5 U / μl） 2×PrimeSTAR ® GC Buffer (Mg 2+plus)* dNTP Mixture（2.5 mM each） 100 μl 1.7 ml×3 800 μl 2. PCR 反应条件*。

Code No. RR047A 研究用PrimeScript TM RT reagent Kitwith gDNA Eraser(Perfect Real Time)说明书目录内容页码●制品说明1●制品内容 1 ●试剂盒外必备材料 1 ●保存 1 ●特长 2 ●使用注意 2 ●操作方法 2 ●Real Time PCR 4 ●实验例 6 ●附录7 ●关联产品8●制品说明为了准确地进行基因表达量分析，必须满足只有cDNA作为模板检出的先决条件，但Total RNA中常常混有基因组DNA，并可以直接作为PCR反应的模板进行扩增，因此会造成解析结果不准确。

为了避免这种情况发生，通常将检测用引物设计在内含子前后的外显子上，使基因组DNA得不到扩增。

但是，此方法不适合具有单个外显子的基因或两个外显子之间所跨的内含子过小的基因，同时当基因组上有伪基因存在时、或设计引物对基因组有非特异性扩增时、以及基因信息没被完全解析的生物种等也同样不适合于本方法。

在这种情况下，我们常常需要对Total RNA样品进行DNase I处理，以除去残存的基因组DNA。

而DNase I处理通常要进行复杂的纯化操作，同时会造成RNA的降解和损失。

PrimeScript RT reagent Kit with gDNA Eraser是可以除去基因组DNA进行Real Time RT-PCR反应的专用反转录试剂。

Kit中使用了具有较强DNA分解活性的gDNA Eraser，通过42℃，2 min即可除去基因组DNA。

同时由于反转录试剂中含有抑制DNA分解酶活性的组分，经过gDNA Eraser处理后的样品可以直接进行15 min的反转录反应合成cDNA，因此，20 min内即可迅速完成从基因组DNA去除到cDNA 合成的全过程。

使用本制品合成的cDNA适用于SYBR® Green分析法和TaqMan®探针分析法，可以根据实验目的，选择与SYBR®Premix Ex Taq II（Tli RNaseH Plus）、Premix Ex Taq（Probe qPCR）等定量试剂组合使用。

Cat. #9790A9791A Product ManualTaKaRa BioMasher Standard (Non-sterile)TaKaRa BioMasher Standard (Sterile)For Research UseTable of ContentsI. Description (3)II. Components (3)III. Storage (3)IV. Materials (3)V. Protocol (3)VI. Experimental Examples (4)VII. Related Products (4)I. DescriptionTaKaRa BioMasher Standard is a disposable microtube homogenizer designed to efficiently crush small amounts of biological samples for nucleic acid and/or protein extraction. This product is a set of a 1.5-ml microtubes and micro stir bars. The inner wall of the tube and the tip of the stir bar are textured allowing effective disruption of the sample. In addition, there is a lid to prevent splashing of the sample and reagents during sample processing. After the sample has been homogenized, the tube can be centrifuged to minimize sample loss.The TaKaRa BioMasher Standard is available either sterilized (Cat. #9791A) or nonsterilized (Cat.# 9790A).II. Components(1) TaKaRa BioMasher Standard (micro stir bar and microtube) 50(2) Stir bar grip (PESTLE GRIP) 1III. Storage Room temperatureKeep out of sun- and UV light.IV. MaterialsTaKaRa BioMasher StandardPolypropylene microtubes AutoclavablePolyacetal stir bar Not autoclavable*Silicon rubber stir bar grip Autoclavable* : The stir bar can not be autoclaved. If you require sterilization, please use Cat. #9791A. V. ProtocolNOTE: Wear a mask, gloves, lab coat, and safety goggles. If necessary, perform in a safety cabinet.1. Insert the micro stir bar into the end of the PESTLE GRIP.2. Add the sample to the microtube. Use less than 100 mg of sample. If necessary, add theextraction reagent (less than 250 μl).3. Insert the micro stir bar into the microtube. With the PESTLE GRIP, rotate and move themicro stir bar up and down, crushing the sample. If necessary, perform on ice.4. After crushing, disconnect the PESTLE GRIP and discard the micro stir bar. The PESTLEGRIP can be used repeatedly; store for future use.5. Close the lid of the microtube. The sample is ready for extraction.VII. Related ProductsRNAisoPlus （Cat. #9108/9109）*NucleoSpin® RNA II （Cat. #740955.10/.50/.250）NucleoSpin® RNA XS （Cat. #740902.10/.50/.250）NucleoSpin® Tissue （Cat. #740952.10/.50/.250）NucleoSpin® Tissue XS （Cat. #740901.10/.50/.250）Wide-Range DNA Ladder (100 - 2,000 bp) （Cat. #3427A/B）* : Not available in all geographic locations. Check for availability in your region.VI. Experimental ExampleTotal RNA extraction from mouse liver 100 mg of mouse liver was crushed using the TaKaRa BioMasher Standard (Sterile). As a comparison, 100 mg frozen mouse liver tissue was crushed with a mortar or with crusher beads. For all samples, total RNA was extracted using 1 ml RNAiso Plus (Cat. #9108/9109) according to the recommended protocol. Total RNA was quantified and analyzed on an 1% agarose gel.Results: Processing the samples using the TaKaRa BioMasher Standard had similar extraction efficiency as the mortar and bead crushing protocols.Crushing method total RNA (μg) A 260/A 2801．Mortar 360 1.702．Mortar 424 1.803．Crusher beads 530 1.924．Crusher beads 494 1.935．TaKaRa BioMasher Standard 411 1.896．TaKaRa BioMasher Standard4311.90M 125364(bp)2,000750Lane 1-6 Equal amounts of RNA M Wide-Range DNA Ladder (100 - 2,000 bp)NOTE : This product is for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals. Also, do not use this product as food, cosmetic, or household item, etc.Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC.If you require licenses for other use, please contact us by phone at +81 77 543 7247 or from our website at .Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements.All trademarks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions.。

PrimeSTAR ® HSDNA Polymerase with GC Buffer使 用 说 明 书TaKaRa Code：DR044A●包装量： 250 U●制品说明PrimeSTAR ® HS DNA Polymerase with GC Buffer适用于高保真扩增具有复杂二级结构的DNA模板（GC rich、重复序列等）。

PrimeSTAR ® HS DNA Polymerase具有极强的3’→5’Exonuclease活性，显示出超群的校正功能，同时还具有优于Taq DNA Polymerase的高扩增效率。

本制品对cDNA克隆等要求高保真的PCR反应能够发挥强大威力,并能对高GC含量模板进行高效扩增。

使用本制品扩增得到的PCR产物几乎都为平滑末端，经磷酸化后可直接克隆于具有平滑末端的载体中。

● 制品内容* Mg 2+浓度为2 mM。

●保 存 -20℃。

●活性定义用活性化的大马哈鱼精子DNA 作为模板/引物，在74℃，30分钟内，摄入10 nmol 的全核苷酸为酸性不溶物的活性定义为1个活性单位（U）。

●纯 度1）10 U 的本酶和0.6 μg 的λDNA-Hin d III 在74℃下反应1小时，DNA 的电泳谱带不发生变化。

2）10 U 的本酶和0.6 μg 的SupercoiledpBR322 DNA 在74℃下反应1小时，DNA 的电泳谱带不发生变化。

●用 途PCR 法高保真扩增DNA 片段。

●PCR 反应性能1） 以λDNA 为模板，可以很好地扩增8、10、12、15 kbp 的DNA 片段。

2） 以人基因组DNA 为模板，可以很好地扩增APO E 基因520 bp（GC 含量74.8%）的DNA 片段。

●反应条件 1. 按下列组份配制PCR 反应液。

2×PrimeSTAR ® GC BufferdNTP Mixture（2.5 mM each）Primer 1 Primer 2Template*PrimeSTAR ® HS DNA Polymerase（2.5 U/μl） dH 2O*【50 μl PCR 反应体系中模板DNA 推荐使用量】人基因组DNA 大肠杆菌基因组DNA λDNA 质粒DNA cDNA Library25 μl4 μl 0.2-0.3 μM （final conc.） 0.2-0.3 μM（final conc.）<200 ng0.5 μlPrimeSTAR ® HS DNA Polymerase（2.5 U / μl） 2×PrimeSTAR ® GC Buffer (Mg 2+plus)* dNTP Mixture（2.5 mM each） 100 μl 1.7 ml×3 800 μl 2. PCR 反应条件*。

TaKaRa Ex Taq®使 用 说 明 书TaKaRa Code ：DRR100A●包装量：250 U●制品说明本制品是应用LA PCR 原理研制的具有3′→5′Exonuclease 活性（Proof reading 活性）的耐热性DNA 聚合酶。

在普通PCR 条件下，与Taq DNA Polymerase 相比，具有扩增效率高、错配率低的优良性能。

使用本制品扩增得到的PCR 产物3′端附有一个“A”碱基，因此可直接克隆于T-Vector 中。

●制品内容74℃，30分钟内，摄入10 nmol 的全核苷酸为酸性不溶物的活性定义为1个活性单位（U）。

●纯 度1） 10 U 的本酶和0.6 μg 的λDNA-Hin d III 在74℃下反应1小时，DNA 的电泳谱带不发生变化。

2） 10 U 的本酶和0.6 μg 的SupercoiledpBR322 DNA 在74℃下反应1小时，DNA的电泳谱带不发生变化。

3）10 U 的本酶和0.6 μg 的λDNA 在74℃下反应1小时，DNA 的电泳谱带不发生变化。

●用 途1）PCR 法扩增DNA。

2）DNA 序列测定。

●PCR 反应性能1） 以λDNA 为模板，可以很好地扩增20 kbp 的DNA 片段。

2） 以人基因组DNA 为模板，可很好地扩增17.5kbp（β-Globin gene）的DNA 片段。

*【50 μl PCR 反应体系中模板DNA 推荐使用量】人基因组DNA 0.1 μg～1 μg 大肠杆菌基因组DNA 10 ng～100 ng λDNA 0.5 ng～5 ng 质粒DNA0.1 ng～10 ng2. PCR 反应条件。

以λDNA 为模板，扩增1 kbp、20 kbp 的DNA 片段的PCR 反应条件如下： 【1 kbp】 94℃ 30 sec.55℃ 30 sec. 30 Cycles 72℃1 min.【20 kbp】 94℃ 1 min. 98℃ 10 sec.68℃ 15 min.72℃10 min.注） PCR 反应条件视模板、引物等的结构条件不同而各异。

JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/biotech_osaka@toyobo.jp1 F0934KKOD -Plus-KOD-201 200 U 200 reactionsStore at -20°C Contents[1]Introduction[2]Components[3]Quality testing[4]Primer design[5]Cloning of PCR products[6]Protocol1. Standard reaction setup2. Cycling conditions[7]Examples[8]Troubleshooting[9] References[10] Related productsC AUSIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe general laboratory precaution and utilize safety while using this kit.JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/biotech_osaka@toyobo.jp 1[ 1 ] Introduction[ 2 ] Components [ 3 ] Quality Testing DescriptionKOD -Plus- is based on DNA polymerase from the hyperthermophilic Archaeon Thermococcus kodakaraensis KOD11) 2). KOD -Plus- exhibits excellent high PCR fidelity and efficiency. The enzyme solution of KOD -Plus- contains two types of anti-KOD DNA polymerase antibodies that inhibit polymerase and 3’J5’ exonuclease activity, thus allowing for Hot Start PCR3). KOD -Plus- generates blunt-end PCR products, due to 3’J5’ exonuclease (proof-reading) activity.Features-Hot Start technology, using anti-KOD DNA polymerase antibodies, results in highly efficient amplification (see Example 1).-KOD -Plus- enables the following amplifications (maximum): 21 kb from lambda phage DNA, 12 kb from human genomic DNA, and 7 kb from cDNA.-KOD DNA polymerase has strong 3’J5’ exonuclease (proof-reading) activity. The PCR error ratio of KOD -Plus- is approx. 80 times less than Taq DNA polymerase.Table. 1 PCR fidelity comparison of each PCR enzyme.*PCR fidelity was based on the mutation frequency of PCR products using a positive-selection base assay with the rpsL gene 4).This reagent includes the following components for 200 reactions:KOD -Plus- (1.0 U/μl) * 200 μl × 110× Buffer for KOD -Plus- 1.0 ml × 125 mM MgSO4 1.0 ml × 12 mM dNTPs 1.0 ml × 1*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize polymerase and 3’J5’ exonuclease activity.Quality check can be performed by amplifying the β-globin gene (3.6 Kb) and p53 gene (4.0 Kb).JAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/biotech_osaka@toyobo.jp 2[ 4 ] Primer Design[ 5 ] Cloning ofPCR products [ 6 ] Protocol -Primers should be 22-34 bases with a melting temperature (Tm) over 60°C. For amplification of a long target, 25-34 bases with high Tm values (≥ 65°C) are recommended. PCR primers should be designed according to the general guidelines.-KOD-Plus- generates blunt-end PCR products, due to 3’J5’ exonuclease (proof- reading) activity. Therefore, the product can be cloned according to a blunt-end cloning method.-PCR products of KOD-Plus- should be purified prior to restriction enzyme treatments. The 3’J5’ exonuclease activity of KOD DNA polymerase remains after the PCR cycles.1. Standard reaction setupThe following procedure is designed for use with the components provided in this kit. Before preparing mixture, all components should be completely thawed, except for the enzyme solution.* Do not use dNTPs from other kits or companies.Notes:-For PCR reactions, thin-wall tubes are recommended. A total reaction volume of 50 μl is also recommended.-The addition of DMSO (final conc. 2-5%) might be effective for amplification of GC-rich targets. Decreased PCR fidelity has been confirmed to not take place with DMSO.-Contaminated RNA (used for cDNA) or genomic DNA inhibits the PCR reaction by chelating Mg2+. PCR should be performed using template DNA containing <100 ng RNA component.Component Volume FinalConcentration 10x Buffer for KOD -Plus- 5 μl 1x2mM dNTPs* 5 μl 0.2 mM each25mM MgSO4 2μl 1.0mM10pmol/μl Primer #1 1.5 μl 0.3 μM10pmol/μl Primer #2 1.5 μl 0.3 μMTemplate DNA X μlGenomic DNA 10-200 ng/50 μlPlasmid DNA 1-50 ng/50 μlcDNA ≤ 100 ng （RNA equiv.)/50 μl PCR grade water Y μlKOD-Plus- (1.0 U/μl) 1μl 1.0 U / 50 μlTotal reaction volume 50 μlJAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/biotech_osaka@toyobo.jp32. Cycling conditionsThe following cycling steps are recommended.Note : If the Tm value of the primer is under 73 °C, the 3-step cycle is recommended.*Tm value of the primer minus 5°C-10°CNotes:-Extension time should be set to 1min per 1 kb of target length.< 2-step cycle >Pre-denaturation: 94 °C , 2 min. Denaturation: 94 °C, 15 sec. Extension:68 °C, 1 min./kb< 3-step cycle >Pre-denaturation: 94 °C, 2 min. Denaturation: 94 °C, 15 sec.Annealing: Tm-[5-10]oC*, 30 sec.Extension:68 °C, 1 min./kb25-35 cyclesJAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/biotech_osaka@toyobo.jp4[ 7 ] ExamplesExample 1．Effect of Hot Start PCR on the generation of primer dimers.Example 2. Effect of addition of DMSO for GC-rich targets.M: 1kb Template: Human genomic DNA Target: TGF-βgene (GC%=70) 2kb Ladder Markers 1: KOD -Plus-, 0% DMSO 2: KOD -Plus-, 2% DMSO 3: KOD -Plus-, 5% DMSOM 1 2 3M 123456MJAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/biotech_osaka@toyobo.jp5[ 8 ] Troubleshooting[ 9 ] References1)Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M, and Imanaka T., Appl Environ Microbiol., 63: 4504-10 (1997)2)Hashimoto H, Nishioka M, Fujiwara S, Takagi M, Imanaka T, Inoue T and Kai Y , J Mol Biol ., 306: 469-77 (2001)3)Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem ., 126: 762-8 (1999)4)Fujii S, Akiyama M, Aoki K, Sugaya Y , Higuchi K, Hiraoka M, Miki Y , Saitoh N, Yoshiyama K, Ihara K, Seki M, Ohtsubo E and Maki H, J. Mol. Bio l., 289: 835-850 (1999)SymptomCauseSolutionLower annealing temperature increments to a maximum of Tm-10°C.Cycling conditions are not suitable.Increase the number of cycles by 2-5 cycles. Mg concentration is low Increase the Mg concentration to 1.2-2 mM. High GC content of target sequenceAdd DMSO 2-5%. <See Example 2>Check the quality of primers. Primer is not good.Redesign primers.Check the quality of template DNA. RNA inhibits amplification.No PCR product/low yield Quality and/or quantity of template DNA is not sufficient.Increase the amount of template DNA.Decrease the number of cycles by 2-5 cycles. Cycling condition is not suitable.Use step-down cycling.Check the quality of primers. Primer is not good.Redesign primers.Too much template DNA Reduce the amount of template DNA. Too much Mg Reduce MgSO 4 to 0.8 mM.Smearing/extra bandToo much enzymeReduce enzyme to 0.5-0.8 U/50 μl.Poor TA cloning efficiency PCR products have blunt- ends.Clone the PCR products according to general blunt- end cloning guidelines.JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/biotech_osaka@toyobo.jp6[ 10 ] Related productsProduct namePackage Code No. TArget Clone -Plus- 10 reactions TAK-201 10x A-attachment mix 25 reactions TAK-301 Ligation high Ver.2750 μl(100 reactions)LGK-201TArget Clone -Plus- is a high efficient TA cloning kit. The kit can be applied to the TA cloning of blunt-end PCR products amplified using KOD -Plus- [Code No. KOD-201] or KOD FX [Code No. KFX-101]. The kit contains pTA2 Vector, 2x Ligation Buffer, T4 DNA Ligase and 10x A-attachment Mix.10 x A-attachment mix is a reagent comprising anti-KOD DNA polymerase antibody specific to KOD 3’J 5’ exonuclease activity (proof-reading activity), as well as Taq DNA polymerase, which exhibits terminal transferase activity. PCR products from KOD -Plus- [Code No. KOD-201] and KOD FX [Code No. KFX-101] possess blunt ends due to 3’J 5’ exonuclease activity of the KOD DNA polymerase. The 10 x A-attachment mix allows for PCR products to acquire overhanging dA at the 3’-ends. Products with 3’-dA overhangs can be directly cloned into arbitrary T-vectors using ligation reagents, such as Ligation high Ver.2 [Code No. LGK-201].Fig. Principle of the 10 x A-attachment mixAnti-KOD DNA polymeraseAJAPAN CHINATOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140www.toyobo.co.jp/e/biotech_osaka@toyobo.jp 7NOTICE TO PURCHASER: LIMITED LICENSEUse of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to US Patent No. 4,889,818.The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim (such as the patented 5’ Nuclease Process claims in US Patents Nos. 5,210,015 and 5,487,972), no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.。