Forward n reversre primer Tm difference is 10 degree - (Jan/06/2008 )

Hi Guys,
I m stuck in this wiered situation, the Tm of my forward primer is 70 degree and reverse is 61 . What kind of temperature gradient should I put for my RT PCR.
I have to stick with the primers because its I need the full ORF of gene which I am going to use in transient Transfection.

Any suggestion on this kind of situation.

regards
Vani

-vani.khare-

QUOTE (vani.khare @ Jan 7 2008, 04:33 AM)

Hi Guys,
I m stuck in this wiered situation, the Tm of my forward primer is 70 degree and reverse is 61

. What kind of temperature gradient should I put for my RT PCR.
I have to stick with the primers because its I need the full ORF of gene which I am going to use in transient Transfection.

Any suggestion on this kind of situation.

regards
Vani

The easiest thing is to extend the reverse primer by a couple of bases. 10 degrees difference is probably too wide for good, specific amplification.

-swanny-