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[Bio101] Standard PCR Protocol   

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In this protocol

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/

Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: He, F. (2011). Standard PCR Protocol. Bio-protocol Bio101: e53. DOI: 10.21769/BioProtoc.53.
Q&A

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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
Department of Biology, University of Pennsylvania, USA

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
Department of Biology, University of Pennsylvania, USA

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
Department of Biology, University of Pennsylvania, USA

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
Department of Biology, University of Pennsylvania, USA

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
Department of Biology, University of Pennsylvania, USA

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

8/31/2011 3:50:44 PM