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RT-PCR (Reverse Transcription- Polymerase Chain Reaction)

RT-PCR (Reverse Transcription- Polymerase Chain Reaction). 操 作. ( 一 ) 细胞总 RNA 的提取. 1 、取小鼠肝脏约 0.1g (约绿豆大小),置匀浆器中,加入 1ml TRIzol ,充分匀浆。 2 、将匀浆后样品转入 Ep 管,离心数秒,取 250ul 上清至一新 Ep 管中。 3 、每管样品中加入 50ul 氯仿(萃取酚),充分振荡混匀,静置 10min 。 4 、 12000rpm×10min 。 5 、取上清转入新 Epg 管,加等体积异丙醇,混匀,室温放置 10min 。

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RT-PCR (Reverse Transcription- Polymerase Chain Reaction)

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  1. RT-PCR(Reverse Transcription-Polymerase Chain Reaction)

  2. 操 作 (一)细胞总RNA的提取 1、取小鼠肝脏约0.1g(约绿豆大小),置匀浆器中,加入1ml TRIzol,充分匀浆。 2、将匀浆后样品转入Ep管,离心数秒,取250ul上清至一新Ep管中。 3、每管样品中加入50ul氯仿(萃取酚),充分振荡混匀,静置10min。 4、12000rpm×10min。 5、取上清转入新Epg管,加等体积异丙醇,混匀,室温放置10min。 6、12000rpm×10min。 7、弃上清,加入50ul 70%乙醇洗涤一次,自然晾干。 8、加入50ul DEPC水溶解沉淀。

  3. (二)逆转录反应(总体积20ul ) RNA (1~4ug) 2 ul Oligo dT 1 ul DEPC处理的ddH2O 5 ul 70℃反应5分钟,迅速置于冰上5分钟 5×buffer 4 ul 10mM dNTP 4 ul 42℃预热5分钟 逆转录酶AMV2 ul RNasin 2 ul 42℃水浴1小时 72℃水浴10分钟 冰上放置备用

  4. (三)PCR扩增(总体积30ul ) ddH2O 15.5 ul 10×Buffer 3 ul Mgcl2 (1.5mmol/L) 2.5 ul dNTPs (各200umol/L) 3 ul Primer 1 (10~100pmol) 1 ul Primer 2 (10~100pmol) 1 ul DNA 模板 (0.1~2ug) 3 ul Taq DNA聚合酶(5U/ul) 1 ul 总体积 30 ul (四)PCR产物电泳分析

  5. PCR(Polymerase Chain Reaction)

  6. 1. What is PCR? • PCR is an in vitro method of DNA synthesis by which a particular fragment of DNA can be specifically amplified. 特异DNA片段的体外快速大量扩增技术.

  7. ——PCR技术的发明者——

  8. 2 Background knowledge of PCR: • DNA replication • DNA denaturation, renaturation & hybridization

  9. DNA Replication:

  10. 3’ 5’ DNA-pol 5’ dCTP dATP dTTP dGTP dCTP dATP dGTP dTTP DNA Replication: Template 3’ 3’ Primer

  11. DNA Denaturation & Renaturation: Denaturation Renaturation heating annealing

  12. Hybridization

  13. 3 Principles of PCR • 高温变性 • 低温退火 • 适温延伸 • 循环25-30次 Denaturation 95℃ Annealing 60℃ Elongation 72 ℃

  14. 5 5 Template DNA Primer 1 5 5 5 5 Primer 2 The 1st cycle 5 5 Denaturation 95℃ Annealing 60℃ Elongation 72 ℃

  15. 5 5 5 5 5 5 Template DNA Primer 1 5 5 5 5 Primer 2 The 1st cycle 5 5 The 2nd cycle 5 5 5 5

  16. 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 The 3th cycle After 20 ~ 30 cycles, the original DNA can be amplified millions-fold.

  17. 指数扩增期 平台期 到达平台期所需PCR循环次数取决于模板拷贝数、PCR扩增效率及DNA聚合酶的种类及活性.

  18. 4 The Characteristics of PCR • High specificity • High sensitivity • Simple and rapid operation • Wide applicability

  19. HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV HBV High specificity HCV HDV HAV HBV PCR

  20. 引物设计的原则 • 引物长度:15-30bp • 引物中碱基分布:尽可能随机分布,G+C占45%~55% • 引物自身不能互补 • 引物之间不能互补 • 引物与非扩增区同源性<70%,3‘末端连续8个碱基不能与待扩增区以外序列完全互补 • 引物3‘端碱基一定要与模板互补,最好选择G和C • 引物5‘端可游离十几个碱基,可进行修饰

  21. High sensitivity Target DNA DNA PCR

  22. PCR仪器设备: • Simple and rapid operation • Wide applicability • 自动移液器和枪头 • 微量离心管 • PCR仪 • 电泳设备

  23. 5 The Composition of PCR System: • template (ng) • primers (0.1-0.5umol/L) • Taq DNA pol (2.5-5U) • dNTP (20-200umol/L) • Mg2+ (1.5-2.5 mmol/L)

  24. Target Sequence DNA Primer 2 Primer 1

  25. DNA聚合酶(DNA polymerase) • 大肠杆菌DNA聚合酶Ⅰ的Klenow片段; • 耐热DNA聚合酶 ?

  26. 耐热DNA聚合酶(Taq DNA pol),来自Thermus aquaticus, 在95℃的半衰期为 1.6 小时。 美国黄石国家公园

  27. 6. Standard PCR reaction: • design the reaction system Target gene : β-actin Primer 1: 5’ATGGGTCAGAAGGACTCCTATC 3’ Primer 2: 5’ATCTCCTGCTCGAAGTCTAGAG 3’ The length of DNA fragment: 530bp

  28. design the reaction system & transfer the reagents in reaction tube ddH2O 15.5 ul 10×Buffer 3 ul Mgcl2 (1.5mmol/L) 2.5 ul dNTPs (各200umol/L) 3 ul Primer 1 (10~100pmol) 1 ul Primer 2 (10~100pmol) 1 ul DNA 模板 (0.1~2ug) 3 ul Taq DNA聚合酶(5U/ul) 1 ul 总体积 30 ul

  29. reaction procedures Heat for 5 min at 95℃ to make the template DNA denaturated completely. Cycle parameters: 95 ℃ 50 sec 60 ℃ 45 sec 72 ℃ 50 sec Cycle times: 30 Finally, incubate for 10 min at 72 ℃ to make all DNA fragments polymerize completely.

  30. detect the amplified DNA molecule by gel electrophoresis

  31. 几种重要的PCR衍生技术 (一)反转录PCR技术 (二)原位PCR技术 (三)实时PCR技术

  32. 实时PCR技术原理

  33. PCR的主要用途 (一)目的基因的克隆 (二)基因的体外突变 (三)DNA和RNA的微量分析 (四)DNA序列测定 (五)基因突变分析

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