PCR amplification - (Apr/24/2006 )
iam getting problem with amplification of genomic DNA.the gene size is around 250bp.i have designed primers to amplify the gene with restriction enzymes Bgl II and Bst EII. both the primers are about 40 nucleotide long.will it be a problem in amplifying the gene?is it ok if the primers are too long.
the primers are having gc % of 55 and tm of >75oC.
i tried annealing temp of 64, 66, 68, 72, 74.
i got little bit amplification around 200bp in all the annealing temp after 40 cycles. but it was very faint band.
and i got one more band around 500bp at annealing temp of 72 and 74.
with increasing temp i should not get non specific bands right?
please suggest me if any body have an idea how to proceed.
thanking you in advance.
here, read through this thread
I suspect you will need a lower Ta for several initial cycles; read through this and you will see why
Try to add 3 to 5% of DMSO in your PCR tubes, it works very well.