ABI StepOne Plus

Takara Code ：DRR390APremix Ex Taq TM(Probe qPCR) （200次量）宝生物工程(大连)有限公司目录内容页码●制品说明1●制品内容 1 ●适用的Real Time PCR扩增仪1●保存 1 ●试剂盒原理 1 ●试剂盒特长 2 ●操作注意 2 ●操作方法 2 ◆应用Thermal Cycler Dice Real Time System扩增仪的操作方法2◆应用ABI PRISM 7000/7700/7300 Real-Time PCR System，StepOnePlus Real-Time PCR System的操作方法4◆应用ABI PRISM 7500/7500 Fast Real-Time PCR System的操作方法 5 ◆应用LightCycler Real Time PCR扩增仪的操作方法7◆应用Smart Cycler II System Real Time PCR扩增仪的操作方法8 ●PCR反应条件说明9 ●进行RT-PCR反应时的实验方法9 ●引物设计说明10 ●特别提示：本公司提供用于基因表达定量分析的引物设计及合成服务11 ●问答11●制品说明本制品是采用探针法（TaqMan®，Molecular Beacon等）进行Real Time PCR（qPCR）反应的专用试剂。

是一种2×浓度的Premix Type试剂，进行实验时，PCR反应液的配制十分方便简单。

Mix中添加了Tli RNaseH（耐热性RNaseH），以cDNA作为模板进行PCR反应时，可以最大限度抑制由于cDNA中残存mRNA对PCR反应造成的阻害作用。

制品中的DNA聚合酶使用了改良后的Hot Start法用DNA聚合酶TaKaRa Ex Taq HS，与Takara精心研制的Real Time PCR用Buffer组合使用，可以有效抑制非特异性的PCR扩增，大大提高PCR的扩增效率，进行高灵敏度的Real Time PCR扩增反应。

Applied Biosystems StepOne™Real-Time PCR System安装标准流程第一部分：安装准备条件ABI StepOne™ Real Time PCR 安装前检查清单尊敬的用户：感谢您购买和使用本公司产品！StepOne™实时定量 PCR 仪是高精密仪器，为保证仪器正常调试及运行，请仔细阅读本说明，在仪器到货前认真准备实验室，并在完成准备工作后将回执传真至 ABI 公司。

谢谢！如果您对文件中的内容有任何不明白之处，请务必与本公司联系，以便本公司相关工程师可就其中要求做进一步沟通。

确认后请在□内打勾√实验室空间条件□的位置。

电脑离StepOne™主机不超过2 米。

实验室通风条件□仪器热量输出约为451.3 瓦，实验室满足通风条件。

实验室环境条件□仪器仅限室内使用，海拔不超过2 千米。

实验室温度可维持在10~30℃，24 小时内温差小于15℃，湿度可维持在15~80%。

建议安装冷暖空调。

实验室电源条件□实验室电源电压为220±10VAC，50Hz，可供电流不小于5A，电源接地良好，即零地电压<3V（或地线阻抗<4Ω）。

该条件非常重要，请以电压表实际测量结果为准。

□为保障仪器安全平稳运行，建议配备一台UPS 或稳压电源。

关于UPS，可以起到稳定电压、防止突然断电和消除电源脉冲干扰的作用。

UPS 型号为在线式，输出波型应为正弦波，功率为1.5-KVA/2 小时以上。

具体品牌建议用户购买“山特牌”，可到其网站查询供应商的联系方法；关于稳压电源，仅可以起到稳定电压的作用，功率为1.5-KVA以上。

□请用户在ABI 工程师到达之前先行安装并调试好UPS，并确保包括地线在内的接线正确。

仪器到货□仪器已到货。

打开标有红色“Open NOW”字样的箱子□到货后立即打开标有红色“Open NOW”字样的箱子。

□此箱中至少包括：TaqMan® RNase P Fast Instrument Verification Plate安装时将需要该试剂。

ABI StepOne/StepOnePlus实时荧光定量PCR系统美国应用生物系统公司是全球生命科学实验解决方案的业界领导者，现在推出了最新采用创新性技术的实时PCR扩增仪——StepOneTM（48孔）和StepOnePlusTM（96孔）实时PCR扩增仪。

这种实时PCR仪操作极其简单，它专门设计了易于使用、但功能强大的用户界面，非常适合不同经验的研究人员使用。

•性价比极高的三色/48孔（StepOne）或四色/96孔（StepOnePlus）系统，可以提供高精度的实时定量PCR结果•基于使用寿命长的LED的光学系统，可以检测多种荧光染料，用于基因表达分析、病原体定量检测、SNP（单核苷酸多态性）基因分型、以及阴性/阳性分析等多种实验•可以进行标准模式和快速模式PCR反应，快速模式可以在40分钟内完成PCR反应•极其紧凑的仪器设计，适合任何大小的实验室环境•LCD触摸屏和支持USB存储，使得系统配置更灵活，并且可以实现无PC操作•便捷的远程监控和电子邮件通知功能，可以节省宝贵的时间•具有独特的VeriFlexTM加热块，它具有六个可独立控制的Peltier加热块，极大地提高了系统的功能和温控的精度•StepOne系统可以升级到StepOnePlus系统，以满足不断提升的研究需要StepOne Software v2.0操作简便、但功能强大的定量PCR分析工具•实验设计向导，帮助您设计和设置实验•提供移液方案和配方，便于快速设置实验•高级设置，适用于需要更多灵活性以便进行更多复杂应用（如多重反应）的专家级的用户•快速启动设定，可以立即启动实验，并在稍后的时间再输入反应板信息•实时监控扩增曲线的增长，便于即时查看实验进程（可通过远程计算机进行监控）•自动基线和自动阈值选定功能，可以简化分析步骤•多图谱视图，可以同时观察四个视图评估数据•自动SNP（单核苷酸多态性）基因分型，具有直观的图表输出和质量评估•故障排除标记，帮助您诊断并解决实验中出现的问题•工具提示功能，当查看扩增曲线或是SNP分型的时候，方便识别不同样本孔•电子邮件通知功能，在实验开始或是结束的时候提醒您•便捷的剪切和粘贴功能•方便地导出为PowerPoint?、Excel?或.jpeg格式的文件7300型实时荧光定量PCR系统。

小学上册英语第3单元综合卷英语试题一、综合题(本题有100小题，每小题1分，共100分.每小题不选、错误，均不给分)1.In a solution, the substance present in the greatest amount is called the ________.2. A ________ is a large, flat area of grassland.3.Fruits provide essential ______ for our bodies. (水果为我们的身体提供必需的营养。

)4.I like to _____ my lunch at school. (eat)anic compounds contain _______ atoms.6.What is the largest mammal in the world?A. ElephantB. Blue WhaleC. GiraffeD. RhinocerosB7.She has a beautiful __________.8.She is a good ________.9.He is playing with his ___. (toys)10.The main component of hemoglobin is _____.11.Which animal is known for its long neck?A. ElephantB. GiraffeC. ZebraD. Kangaroo12.The United Kingdom is made up of England, Scotland, Wales, and ________.13.The rabbit hops _____ (quickly/slowly).14.What is the largest organ in the human body?A. HeartB. LiverC. SkinD. Brain15.I see a _____ (mouse) in my house.16.The ________ is a colorful bird that sings.17.The _______ (犬) is often considered man's best friend.18.I have a lot of fun with my toy ____ set. (玩具名称)19.The __________ is perfect for a picnic in the park. (天气)20. A ______ (骆驼) can survive without water for days.21.The __________ (葡萄牙) was a major player in the Age of Exploration.22.What do we call the study of heredity and variation?A. GeneticsB. EvolutionC. BiologyD. Ecology23.What is the capital city of Italy?A. RomeB. FlorenceC. VeniceD. MilanA24. A solution that does not conduct electricity is called a _______.25.The monkey swings from ______ to ______.26.What do we use to write?A. BrushB. PencilC. SpoonD. ForkB27.The _____ (草地) is perfect for playing outside.28.I enjoy making my own games using my ________ (玩具名). It sparks my imagination.29.The ________ Revolution began in the late 18th century.30.My ________ (玩具名称) can change shapes easily.31.The children are ___ in the park. (running)32.The octopus can taste with its ______ (触手).33.What animal is known as “man's best friend”?A. CatB. DogC. RabbitD. Hamster34.Cosmic rays are high-energy particles that travel through ______.35.The capital of Samoa is __________.36. A _____ (种子库) preserves different types of seeds.37.What is the opposite of "cold"?A. CoolB. WarmC. HotD. FreezingC38.I have a toy _______ that can spin and twirl around.39.I enjoy drawing and painting in my free time.40.What is the name of the largest freshwater lake in the world?A. Lake SuperiorB. Lake VictoriaC. Caspian SeaD. Lake BaikalD41.The capital of Russia is _____ (51).42.My sister loves to _______ (动词) in the park. 我们常常一起 _______ (动词).43.The ______ teaches us about famous historical figures.44.I enjoy ______ with my family.45.__________ (测量工具) are vital for accurate chemical analysis.46.The __________ (Renaissance) was a period of great cultural revival in Europe.47.The ancient Egyptians used _______ to write on. (纸草)48.The chemical formula for silver nitrate is ______.49.My mom cooks _______ (美味的) food.50. A shooting star is also called a ______.51. A __________ is a natural phenomenon that can impact ecosystems.52.What do we call the outer layer of the Earth?A. CrustB. CoreC. MantleD. LithosphereA53.The ant is strong for its _________ (大小).54.What is the capital city of the USA?A. New YorkB. WashingtonC. Los AngelesD. ChicagoB55.My dream is to have a room full of ____. (玩具名称)56.What is the name of the famous wizard in "Harry Potter"?A. DumbledoreB. GandalfC. MerlinD. HagridA57.My teacher encouraged us to create our own ________ (漫画). I drew a funny ________ (角色).58.The _______ of sound can be affected by the distance from the source.59. A strong acid has a pH value that is ________ than60.We can _______ (跑步) fast.61.What is the capital of Peru?A. LimaB. QuitoC. BogotáD. SantiagoA62.Emma is a ______. She likes to read stories.63.The capital of Pakistan is __________.64.The _____ (土地) must be prepared before planting.65. A concentrated solution contains a high amount of ______.66.What is the main ingredient in salad?A. MeatB. VegetablesC. BreadD. Cheese67.Hydrogen is the simplest and most abundant _____.68.My _______ (蜗牛) is very slow and steady.69.The _____ (植物组织) is made up of different cells.70.My cousin is a talented ____ (dancer).71.What is the main ingredient in chocolate chip cookies?A. SugarB. FlourC. Chocolate chipsD. Eggs72.What is a comet mostly made of?A. RockB. MetalC. Ice and dustD. Gas73. A puppy loves to play with ______ (球).74.I read a _____ (书) every night.75.What is the name of the famous statue in New York Harbor?A. Christ the RedeemerB. Statue of LibertyC. DavidD. Venus de MiloB76.What is the opposite of "happy"?A. SadB. ExcitedC. JoyfulD. AngryA77.The _____ (自然资源) includes all the plants and animals.78.The _____ (watermelon) is refreshing.79.Did you see that _____ (小狗) rolling in the grass?80.My brother has a deep love for __________ (自然).81.The molecule responsible for carrying oxygen in the blood is ______.82. A __________ has a flat body and can often be found in rivers.83.We _____ (play/plays) games on weekends.84. A force can change an object’s ______.85.What is the opposite of "speak"?A. TalkB. WhisperC. SilenceD. ShoutC86.What is the capital city of Australia?A. SydneyB. MelbourneC. CanberraD. Brisbaneets have a bright tail that points away from the _______.88.What do we call an animal that lays eggs?A. MammalB. ReptileC. BirdD. Both B and C89.My cousin is very __________ (适应力强).90. Wall is one of the most famous ________ (地标). The Grea91.What is the name of the famous theme park located in California?A. DisneylandB. Universal StudiosC. SeaWorldD. LegolandA92.What do we call the act of working together toward a common goal?A. CooperationB. CollaborationC. TeamworkD. PartnershipCmunity meeting) discusses local issues. The ____94.I enjoy ______ (与朋友互动).95.The nurse plays a vital role in _____ (患者护理).96.What do you call the written record of events?A. HistoryB. ScienceC. GeographyD. MathA97.How many senses do humans have?A. 4B. 5C. 6D. 7B98.My uncle plays soccer on the ____ (weekends).99. A ____ is a friendly animal that enjoys human companionship. 100. A shadow is formed when light is ______.。

qpcr步骤范围和适用性本实验适用于使用StepOnePlus™实时PCR仪对DNA PE、DNA PE Index、SE文库以及Exon Capture组、转录组、表达谱、Small RNA组、表观遗传组等制备的文库进行上机前的精确定量。

摘要荧光定量PCR实验是以Agilent 2100 Bioanalyzer对文库的检测结果为参照, 使用StepOnePlus™实时PCR仪对文库进行定量,得出待测文库浓度，为之后的Cluster制备提供浓度依据。

该实验的主要内容包括：（1）梯度稀释标准品；（2）按实验要求稀释样品；（3）配制反应预混液；（4）加样；（5）设置反应程序；（6）运行实验；（7）分析实验结果，计算待测样品浓度。

整个流程需要3-4小时。

缩写词和SOP中专业术语的解释缩写词或术语解释基线(Baseline) 扩增曲线中的水平部分阈值(Threshold) 高于基线但处于扩增曲线的指数增长区范围之内的荧光水平。

C t值扩增产物的荧光信号达到设定的阈值时所经过的扩增循环数。

实验材料及方法1 实验试剂和耗材1.1试剂表1-1 实验所需试剂试剂名称货号生产厂家10×Buffer DDR006B TAKARAdNTP(2.5mM) DDR006B TAKARAEX HS Taq DDR006B TAKARA TaqMan PE Probe 上海生工PE Q-PCR Primer 1.1 上海生工Index Q-PCR Primer RR 上海生工SE Q-PCR Primer 1.1 上海生工SE Q-PCR Primer 2.1 上海生工ROX 12223－012 Invitrogen含ThermoPol缓冲液B9004S NEBMgSO4(100mM)Tween20 P9416-100ML SIGMADMSO DT0163-500ML 上海生工Betaine B2629-1KG SIGMASRNA PCR Primer 1 上海生工SRNA PCR Primer 2 上海生工1.2 耗材表1-2 实验所需耗材耗材型号生产厂家96孔国产光学反应板国产光学封口膜（泛特津）国产0.2ml Sterile, PCR microtubes PCR-02D-C Axygen0.6ml离心管MCT-060-C Axygen吸嘴0.5-10ul超微量、袋装T-300 Axygen吸嘴200ul黄色吸嘴、袋装T-200-Y Axygen吸嘴1000ul蓝色、袋装T-1000-Y Axygen1.5ml离心管MCT-150-C Axygen2 实验仪器设备表2-1 实验所需仪器设备设备型号生产厂家StepOne Plus™ Real-Time 4376592 ABI迷你离心机LX-200 海门其林贝Vortex QL－海门其林贝微孔板离心机MPS-10Labnet3 实验操作流程3.1 填写Q-PCR实验登记表样品的交接按照《质控组样品交接管理规程》执行。