PCR常见问题的精辟总结--耶鲁大学

Troubleshooting for PCR and multiplex PCR

Troubleshooting discussion is based on the PCR protocol as described in the table below. All reactions are run for 30 cycles.

COMPONENT VOLUME FINAL CONCENTRATION

1.autoclaved ultra-filtered water (pH 7.0) 20.7µL -

2.10x PCR Buffer* 2.5µL 1x

3.dNTPs mix (25 mM each nucleotide) 0.2µL 200 µM (each nucleotide)

4.primer mix (25 pmoles/µL each primer) 0.4µL 0.4 µM (each primer)

5.Taq DNA polymerase (native enzyme) 0.2µL 1 Unit/25 µL

6.genomic DNA template (100 ng/µL) 1.0µL 100 ng/25 µL

* The 10x PCR buffer contains: 500 mM KCl; 100 mM Tris-HCl (pH 8.3); 15 mM MgCl2 (the final concentrations of these ingredients in the PCR mix are: 50 mM KCl; 10 mM Tris-HCl; 1.5 mM MgCl2).

QUESTIONS SOLUTIONS

1. I get (many) longer unspecific products. What can I do?

Decrease annealing time

Increase annealing temperature

Decrease extension time

Decrease extension temperature to 62-68º C

Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM.

Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.

Take less primer

Take less DNA template

Take less Taq polymerase

If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)

Combine some/all of the above

2. I get (many) shorter unspecific products. What can I do?

Increase annealing temperature

Increase annealing time

Increase extension time

Increase extension temperature to 74-78º C

Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mM

Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant

Take less primer

Take less DNA template

Take less Taq polymerase

If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)

Combine some/all of the above

3. Reaction was working before, but now I can't get any product. Make sure all PCR ingredients are taken in the reaction (buffer, template, Taq, etc)

Change the dNTP solution (very sensitive to cycles of thawing and freezing, especially in multiplex PCR)

If you just bought new primers, check for their reliability (bad primer synthesis ?)

Increase primer amount

Increase template amount

Decrease annealing temperature by 6-10º C and check if you get any product. If you don't, check all your PCR ingredients. If you do get products (including unspecific ones) reaction conditions as described above.

Combine some/all of the above

4. My PCR product is weak. Is there a way to increase the yield? Gradually decrease the annealing temperature to the lowest possible.

Increase the amount of PCR primer

Increase the amount of DNA template

Increase the amount of Taq polymerase

Change buffer (KCl) concentration (higher if product is lower than 1000bp or lower if product is higher than 1000bp)

Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol.

Check primer sequences for mismatches and/or increase the primer length by 5 nucleotides

Combine some/all of the above

5. My two primers have very different melting temperatures (Tm) but I cannot change their locus. What can I do to improve PCR amplification?

An easy solution is to increase the length of the primer with low Tm. If you need to keep the size of the product constant, add a few bases at the 3' end. If size is not a concern, add a few bases at either the 3' or the 5' end of that primer.