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.

* 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 and 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 4º C.
　　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. Unspecific products 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 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
　　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)