Poor PCR Results
Posted 27 July 2001 - 09:00 PM
Our laboratory has spent several months working on genetically modified soybeans and soy products. We have experienced difficulty in getting consistent PCR results of those GM genes. Although we got fairly good PCR products at the beginning, recent PCR results show either a lot of smear with no identifiable bands or only primer dimers (i.e., no PCR products observed). Using new primers and new templates and/or lowering the annealing temp did not solve the problems.
Any suggestions would be deeply appreciated. Thanks.
Posted 27 July 2001 - 09:00 PM
Posted 03 August 2001 - 09:00 PM
1. I get (many) longer unspecific products. What can I do?Decrease annealing timeIncrease annealing temperatureDecrease extension timeDecrease extension temperature to 62-68º CIncrease 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 primerTake less DNA templateTake less Taq polymeraseIf none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)
2. I get (many) shorter unspecific products. What can I do?Increase annealing temperatureIncrease annealing timeIncrease extension timeIncrease extension temperature to 74-78º CDecrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mMIncrease MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constantTake less primerTake less DNA templateTake less Taq polymeraseIf none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s)
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 amountIncrease template amountDecrease 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.
4. My PCR product is weak. Is there a way to increase the yield?Gradually decrease the annealing temperature to the lowest possible (touch down PCR).Increase the amount of PCR primerIncrease the amount of DNA templateIncrease the amount of Taq polymeraseChange 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
5. My two primers have very different melting temperatures 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.