Run PCR amplifications in agarose gels - (May/03/2010 )
I am relatively new in biotopics and you will probably think that my question is so basic, but I would like that someone support my suspicion.
I ran a PCR with primers used in a paper with good results. I tried to repeat their experiment with another cells but I cannot obtain the expected fragment. Instead of the 214bp fragment I obtain a band of approximately 100bp. I know that they are not dimers since they do not appear in the blank (master mix without DNA) and I have studied the secondary structures using bioinformatics tools. So I think that they are unspecific amplifications. After this I want to ask you if there exist some way to study if they really are unespecificities. I have tried to run a BLAST but I obtain no satisfactory results.
Thanks you very much for your help.
What do you mean you have studied the secondary structures?
Why do you want to know what the non-specific product is anyway?
I would just work on optimising your PCR with the new sample.
criscastells on May 3 2010, 04:26 PM said:
What are the primer sequences and species?
You can always sequence your product using your PCR primers to see what it is.
Is it possible, that your cells have a deletion in the region you amplify? How did you know that your fragment should be 214 bp, did you tested your primers on primer3 or something?
When I talk about secondary structures I mean that I have calculate the free energy formation of primer dimers, and they show that there is not dimer formation.
I use primer blast and it corroborate that the fragment amplified must to be of 214bp. I really don't know if it is possible to be a deletion but I think not since they are human dermal normal fibroblast without any special treatment. Anyway I think that my following step will be to optimize the PCR program.