本文章节


 

Standard PCR Protocol
标准PCR方案   

引用 收藏 8次提问与回复 分享您的反馈 被引用

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/
登录/注册账号可免费阅读全文
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: He, F. (2011). Standard PCR Protocol. Bio-101: e53. DOI: 10.21769/BioProtoc.53.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

Ade Bidemi
Akwa Ibom State University
Wow. This is my first time. This platform really is helpful thanks.
2020/8/24 21:20:23 回复
maham khan
uog
its difficult to understand
2014/9/7 11:16:08 回复
sehrish Aslam
kinnaird college

Hi have a look at this article..https://www.scienceofhealthy.com/polymerase-chain-reaction-pcr/
thanks

2019/8/4 12:28:56 回复


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.
2013/10/30 17:52:53 回复
sopheap yun
Kyoungbook National University
Dear, author
I pleasure to know your website.
Today I would like as you. How can do for electrophoresis ?
2013/6/29 7:15:21 回复
Fanglian He
Bio-protocol

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.

2013/7/1 14:36:42 回复


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
2013/1/22 21:07:20 回复
Fanglian He
Bio-protocol

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.

2013/1/23 16:17:34 回复


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?
2012/5/16 21:37:34 回复
Fanglian He
Bio-protocol

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

2012/5/20 14:45:07 回复


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 ?
2011/5/20 12:34:39 回复
Fanglian He
Bio-protocol

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

2011/8/31 15:51:34 回复


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
2011/5/19 12:58:09 回复
Fanglian He
Bio-protocol

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

2011/8/31 15:50:44 回复