PCR from human genomic DNA - (Dec/29/2005 )
I have been trying to get a 800bp PCR product from human genomic DNA, the 5 end primer is on the first exon, and the 3 end primer is on the intron which is very close to the end of the second exon, the PCR conditions is as following:
94oC, 5 minutes
Mg2+ concentration is 1.5mM
DNA template is 100ng
I didn't see any bands come out except the primer dimer. I appreciate if anybody can help me with this? thank you very much.
perhaps you need new primers...but sometimes additives (DMSO, PEG), or adjusting the annealing temp can help with a tricky PCR..have you tried an annealing temp gradient? But if your primers have a high affinity for each other, all this monkeying around may not help. another possibility, add less primer
Thank you, I will try less primer and different temperature.
check the GC content of your template, if it is above 60% try using DMSO in your pcr conc should be 5% final conc of pcr. optimise annealing temp using gradient pcr. should see some bands
As I recently faced similar problem changing dNTPS and water helped me.