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.

6. I have a number of primer pairs I would like to use together. Can I run a multiplex PCR with them?. How?

Very likely, yes.

Try amplify all loci seaprately using the same PCR program. If one of the primer pairs yields unspecific products, keep the cycling conditions constant and

change other parameters as mentioned above (#1 and #2).

Mix equimolar amounts of primers and run the multiplex reaction either in the same cycling conditions or by decreasing only the annealing temperature by 4ºC.

If some of the loci are weak or not amplified, read below !!

7. How many loci can I amplify in multiplex PCR at the same time? 

Difficult to say. The author has routinely amplified from 2 to 14 loci.

Literature describes up to 25 loci or so.

8. One or a few loci in my multiplex reaction are very weak or invisible. How can amplify them?

The first choice should be increasing the amount of primer for the "weak" loci at the same time with decreasing the amount of primer for all loci that can be

amplified. The balance between these amounts is more important than the absolute values used !!.

Check primer sequences for primer-primer interactions

9. Short PCR products in my multiplex reaction are weak. How can I improve their yield?

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

Decrease denaturing time

Decrease annealing time and temperature

Decrease extension time and temperature

Increase amount of primers for the "weak" loci while decreasing the amount for the "strong" loci.

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

Combine some/all of the above

10. Longer PCR products in my multiplex reaction are weak. How can I improve their yield?

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.

Increase denaturing time

Increase annealing time

Decrease annealing temperature

Increase extension time and temperature

Increase amount of primers for the "weak" loci while decreasing the amount for the "strong" loci

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

Combine some/all of the above

11. All products in my multiplex reaction are weak. How can I improve the yield?

Decrease annealing time in small steps (2º C)

Decrease extension temperature to 62-68º C

Increase extension time

Increase template concentration

Increase overall primer concentration

Adjust Taq polymerase concentration

Change KCl (buffer) concentration, but keep MgCl2 concentration at 1.5-2mM

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

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

Combine some/all of the above

12. Unspecificproducts appear in my multiplex reaction. Can I get rid of them somehow?

If long: increase buffer concentration to 1.2-2x, but keep MgCl2 concentration at 1.5-2mM

If short: decrease buffer concentration to 0.7-0.9x, but keep MgCl2 concentration at 1.5-2mM

Gradually increase the annealing temperature

Decrease amount of template

Decrease amount of primer

Decrease amount of enzyme

Increase MgCl2concentration up to 3-4.5 mM but keep dNTP concentration constant

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

If nothing works: run PCR reactions for each (multiplexed) locus individually, using an annealing temperature lower than usual. Compare the unspecific

products for each locus tested with the unspecific products seen when running the multiplex PCR. This may indicate which primer pair yields the unspecific

products in the multiplex reaction.

Combine some/all of the above

(Note: primer-primer interactions in multiplex PCR are usually translated into lack of some amplification products rather than the appearance of unspecific products)