Replies

• Eva Amsen

  10 March 2008 | 21:31

  1. Bands below 100 bp are probably primer dimers, which might be a problem with the design of the primers. What program (if any) are you using to design primers?

  2. Once you get rid of primer dimers, if you still don’t see your expected product, you can try different annealing temperatures. Some PCR machines allow you to set a gradient, where you can put several tubes in at once, and the tubes in one part of the machine will have an annealing temp of (for exampl) 53 degrees and those in another part will have 56 degrees… whatever temperatures you want to look at.

  I don’t have any other suggestions. All my PCRs have always worked with these two troubleshooting steps.

• Joao Goncalves

  11 March 2008 | 11:10

  I agree with Eva, you’re probably having primer dimers there.

  You must follow the basic rules for primer design…like avoiding secondary structures, ending the primer sequence with Cs our Gs (at the 3’ end), etc…

  The gradient PCR will probably solve your problem if the problem is annealing temperature.

  I don’t know the GC content of the sequence you’re trying to amplify but if you have a sequence very rich in A and T the extension temperature should be 60ºC instead of 72ºC (with Taq polymerase) – believe me, it makes a big difference.

  Good luck.