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[Bio101] Standard PCR Protocol
[Bio101] 标准PCR方案   

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Abstract

This protocol describes basic steps of a PCR experiment using home-made Taq DNA polymerase. Some steps may vary with different DNA polymerase.

Materials and Reagents

  1. Tris-HCl (Sigma-Aldrich)
  2. KCl (EM SCIENCE)
  3. MgCl2 (EM SCIENCE)
  4. Gelatin (Sigma-Aldrich)
  5. Taq DNA polymerase (home-made)
  6. dNTPs (New England Biolabs, catalog number: N0447L )
  7. Template DNA (genomic, plasmid, cosmid, bacterial/yeast colony, etc.)
  8. Primers

Equipment

  1. Thermal cycler (MJ Research)

Procedure

  1. Prepare DNA template:
    Usually, for plasmid DNA, 1-10 ng; for genomic DNA, 50-100 ng per reaction is needed. Normally, DNA template does not need to be purified. However, both purity and the amount of template can strongly influence the outcome of the reaction.

  2. Design primer:
    Generally, primers used are 18-23 mer in length. Use Primer3 free online software (reference 1) to design primers.

  3. Determine annealing temperature:
    Melting temperature (Tm) of primers can be calculated by the following formula: Tm = [(#of A + T residues) x 2] + [(#of G + C residues) x 4] °C. Tm-5 °C is a good annealing temperature to start with. However, optimal annealing temperatures can only be determined experimentally for a certain primer/template combination. Temperature gradient PCR is often a way to finalize an optimal annealing temperature.

  4. Prepare 10x PCR reaction buffer, include:
    100 mM Tris-HCl (pH 8.3)
    500 mM KCl
    15 mM MgCl2
    0.1% gelatin
    Note: The MgCl2 concentration is typically 10-15 mM. However, the optimum concentration needs to be determined experimentally. Mg2+ forms a soluble complex with dNTP's which facilitates dNTP incorporation, and stimulates polymerase activity. It also promotes and stabilizes primer and template interaction. Thus, Increasing the magnesium concentration has the same effect as lowering the annealing temperature. Too low Mg2+ leads to low yields (or no yield) and too much Mg2+ cause nonspecific products.

  5. For a 100 μl reaction, add:
    10x PCR buffer 10 μl
    DNA template (5 ng μl-1) 1 μl
    Primer A (50 mM) 1 μl
    Primer B (50 mM) 1 μl
    dNTPs (2 mM) 10 μl
    Taq (5 U μl-1) 1 μl
    Sterile ddH2O 76 μl
    Notes:
    1. For some PCR machines that do not have a heated lid, mineral oil needs to be added to each reaction to prevent evaporation of the sample.
    2. Prepare a control reaction with no template DNA and an additional 10 μl of sterile water.

  6. A typical PCR program may be:
    1. Initial denaturation, 4-8 min at 94-95 °C.
    2. Denaturation, 15 sec at 94-95 °C.
    3. Annealing, 15 sec at x °C (depends on Tm).
    4. Extension, x sec (depends on product length, 1 min kb-1) at 72 °C.
    5. Return to step 2 for 30-35 additional cycles.
    6. Final extension, 10 min at 72 °C.
    7. Keep sample at 4 °C until loading.

References

  1. http://frodo.wi.mit.edu/primer3/

简介

该协议描述了使用自制Taq DNA聚合酶的PCR实验的基本步骤。 一些步骤可随不同的DNA聚合酶而变化。

材料和试剂

  1. Tris-HCl(Sigma-Aldrich)
  2. KCl(EM SCIENCE)
  3. MgCl (EM SCIENCE)
  4. 明胶(Sigma-Aldrich)
  5. Taq DNA聚合酶(自制
  6. dNTP(New England Biolabs,目录号:N0447L)
  7. 模板DNA(基因组,质粒,粘粒,细菌/酵母菌落等)
  8. 引物

设备

  1. 热循环仪(MJ Research)

程序

  1. 准备DNA模板:
    通常,对于质粒DNA, 对于基因组DNA,需要每反应50-100ng。 通常,DNA模板不需要纯化。 然而,纯度和模板量都会强烈影响反应的结果
  2. 设计底稿:
    通常,使用的引物长度为18-23mer。 使用Primer3免费在线软件(参考文献1)设计引物
  3. 确定退火温度:
    引物的熔解温度(Tm)可以通过下式计算:Tm = [(A + T残基)×2] + [(G + C残基)×4]℃。 Tm-5℃是开始的良好退火温度。 然而,最佳退火温度只能对于某种引物/模板组合通过实验确定。 温度梯度PCR通常是最终确定最佳退火温度的方法
  4. 准备10x PCR反应缓冲液,包括:
    100mM Tris-HCl(pH8.3)
    500 mM KCl
    15mM MgCl 2·h/v 0.1%明胶
    注意:MgCl 2 浓度通常为10-15 mM。然而,最佳浓度需要通过实验确定。 Mg 2 + 与dNTP形成可溶性复合物,其促进dNTP掺入并刺激聚合酶活性。它还促进和稳定引物和模板相互作用。因此,增加镁浓度具有与降低退火温度相同的效果。太低的Mg 2 + 导致低产量(或无产量) + 造成非特定产品。

  5. 对于100μl反应,添加:
    10x PCR缓冲液10μl
    DNA模板(5ngμl -1 )1μl
    引物A(50mM)1μl
    引物B(50mM)1μl
    dNTPs(2mM)10μl
    Taq(5Uμl -1 )1微升
    无菌ddH 2 @76μl
    注意:
    1. 对于没有加热盖的一些PCR机,需要向每个反应中加入矿物油以防止样品蒸发。
    2. 准备没有模板DNA和另外10μl无菌水的对照反应。

  6. 典型的PCR程序可以是:
    1. 初始变性,94-95℃下4-8分钟
    2. 变性,在94-95℃下15秒
    3. 退火,在x℃下15秒(取决于Tm)
    4. 延伸,在72℃下x秒(取决于产物长度,1分钟kb -1 )。
    5. 返回步骤2,持续30-35个周期。
    6. 最终延伸,72℃10分钟
    7. 将样品保持在4°C直至装载

参考文献

  1. http://frodo.wi.mit.edu/primer3/

  • English
  • 中文翻译
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:He, F. (2011). Standard PCR Protocol. Bio-protocol Bio101: e53. DOI: 10.21769/BioProtoc.53;
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maham khan
uog
its difficult to understand
9/7/2014 11:16:08 AM Reply
Neha Sharma
HPAU
hello
I am doing the characterization of full genome of a tobamovirus. I am getting the problem in RACE PCR. I have synthesized the cDNA using the protocol given in the RACE manual (SMART? RACE cDNA Amplification Kit
User Manual) i got the amplification for the very first time but now i m not getting the amplification i have repeated PCR three times so there is no room for human error and my chemicals are all good. Can u give me suggestion.
thank u.
10/30/2013 5:52:53 PM Reply
sopheap yun
Kyoungbook National University
Dear, author
I pleasure to know your website.
Today I would like as you. How can do for electrophoresis ?
6/29/2013 7:15:21 AM Reply
Fanglian He
University of Pennsylvania

Please specify which types of gel electrophoresis (for DNA/RNA or protein?) you were asking. Since DNA/RNA gel electrophoresis is relatively simple, we do do not have such detailed protocols on our site. But, you could find many good protocols on internet, such as http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html

Fro protein gel electrophoresis, you may search our website by using keyword "electrophoresis" and see if you could find answers that you are looking for.

7/1/2013 2:36:42 PM


here u include std PCR Protocol but i come to know d annealing temperature for alkaline phosphatase PCR please
mention my query and solve it....THANK YOU
1/22/2013 9:07:20 PM Reply
Fanglian He
University of Pennsylvania

I am not sure I understand your question well. To my knowledge, alkaline phosphatase is used to clean up PCR product by removing excess dNTPs (although I never used alkaline phosphatase for this purpose). So, I assume this step is done after PCR reaction.

1/23/2013 4:17:34 PM


quan jiang
the university of hong kong
"home-made Taq DNA polymerase"
It is delighted reading your protocol regarding home-made Taq Polymerase purification. . I must say that it is good news for a small lab with limited budget. I would like to make my own lab DNA polymerase. Would you send clone to me at your convenience?
5/16/2012 9:37:34 PM Reply
Fanglian He
University of Pennsylvania

You can request Taq polymerase plasmid from Addgene, http://www.addgene.org/25712/.

5/20/2012 2:45:07 PM


Yuanqing Lin
in in PCR tagging process, there is tagging step. so one of it is IMAC. so , how to activate the column to higher the rate of selectivity ?
5/20/2011 12:34:39 PM Reply
Fanglian He
University of Pennsylvania

Please rephrase your question. Which step of the procedure that your question was related to?

8/31/2011 3:51:34 PM


Yuanqing Lin
I have prepared a frsh 10x PCR buffer as described and I had tested it. There was amplification however there is a trailing effect from the well.... So what could be the possible reason for this? And how can I trouble shoot this problem

chaitanya
scholar,
India
5/19/2011 12:58:09 PM Reply
Fanglian He
University of Pennsylvania

I am not sure about what you meant by "a trailing effect from the well".

8/31/2011 3:50:44 PM