PCR on Genomic DNA - (Feb/20/2006 )
I'm trying to do a PCR to get ~800bp from genomic DNA and I get nothing, I've already tried:DMSO, different Tm, different concentration of Mg, primer and DNA (I use a template the once worked with other primers) and also the FailSafe kit from Epicentre
and I get nothing
Any suggestion pleeeeeeeeeaaaasssss !!!!!
Is there any way the I can check that my primers are good
you could try putting them into the PCR facility at UCSC (http://www.genome.ucsc.edu/cgi-bin/hgPcr) - remembering to change the database to mouse or human....
Or you could blast them at NCBI
Did you order your reverse primer the right way round? (an all too common mistake really - even for the most experienced person!!!)
Did you use any programs to help you design them? Might help if you are unsure......
It does sound like a primer problem...
Since you mention you have another set of primers that work on the template, run them as your positive control along with your experiment, using the same conditions as your experiment, to confirm the template, Taq, etc. are not the problem.
What is your PCR profile (temps, number of cycles, etc.)?