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A general PCR protocol and procedure note



A general PCR protocol and procedure note

General PCR protocol:

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10X reaction buffer (with MgCl2) 5 ul
25mM MgCl2(optional) 3 ul
dNTP mix (10mM of each dNTP) 1 ul
Taq DNA Polymerase(5U/ul) 0.25ul
downstream primer (10 um/ul) 1.5ul
upstream primer (10 um/ul) 1.5ul
DMSO 2.5ul
template DNA (10-100ng/ul) 1-2 ul

nuclease-free water (adjust to a final 50 ul)

Cycle condition:

95 C 5 min denature step

25-35 cycles:
95 C 30 seconds denature step
55 C - 65 C 30 seconds - 60 seconds annealing primer with single strand DNA template,
72 C 30 seconds - 60 seconds extension step (time varies depending on the length of amplified DNA)
72 C 8’ final extension step

Note:
False negative due to PCR—In amplification reactions where no product is generated, further testing is recommended to confirm the presence of amplifiable DNA. False negatives are often due to PCR inhibitors present in the specimen, so demonstration that another gene or DNA sequence (different from the one being tested) can be successfully amplified in the same specimen should be sufficient to rule out this artifact.
The best way to avoid cross-contamination is to use the following order of preparation within amplification run: actual samples, followed by positive controls, followed by negative controls.
Pre- and post-amplification samples should be manipulated in physically separate areas.

Procedure:

1. Thaw 10x buffer, dNTPs, and primers. Keep on ice.
2. NOTE: ALWAYS INCLUDE A REACTION TUBE “BLANK” WHICH WILL HAVE ALL INGREDIENTS EXCEPT DNA. THIS IS AN ESSENTIAL CONTROL FOR EACH PCR REACTION.
3. Create a bulk reagent tube for each set of primers to be used. This will contain the 10x buffer, nucleotides, polymerase, primers, and water that will be required for this set of PCR reactions. For the purposes of determining amounts to use in the bulk reagent tube, consider that you will have 11 reactions for each 10 reactions you will be setting up. Label bulk reaction tubes with primers to be used and number or reactions you will be setting up. Mix well and place on ice.
4. Use 0.2 ml thin walled centrifuge tubes for reaction. Label top of tubes with sample #
5. Pipet mix reagent (except template) into each tube.
6. Pipet DNA samples into appropriate tubes, using a new tip for each sample.
7. Mix tube well. Spin for 5 seconds in microfuge to remove liquid from sides of tube.
8. Place tubes in PCR machine, program machine, and begin PCR reaction.

Analysis:

1. Analyze the PCR reaction products by agarose gel electrophoresis of a 5 ul aliquot from the total reaction. The products should be readily visible by UV transillumination of the ethidium bromide-stained gel.
2. Store reaction products at -20 C until needed. The reaction products can be further purified using a number of procedures, including the Qiagen PCR gel extract or column purification system.

References:

1. Molecular Cloning: A Laboratory Manual Second Edition (Maniatis)
2. The Source for Discovery (Promega)
3. PCR protocol with Taq DNA polymerase
4. PCR Success Guidelines
5. General PCR Protocol

Last update 07-Feb-2007, Rating Very Good of 6 votes.

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