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 2. I get (many) shorter unspecific products. What can I do? Increase annealing temperature |
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) 4. My PCR product is weak. Is there a way to increase the yield? Gradually decrease the annealing temperature to the lowest possible. 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. 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. 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 !!. |
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 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 11. All products in my multiplex reaction are weak. How can I improve the yield? Decrease annealing time in small steps (2º C) 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 |
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