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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
94oC, 30'
55oC, 1'30"
72oC, 2'
30 cycles
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.


QUOTE (gap @ Dec 30 2005, 05:25 AM)
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.