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Huge difference in Tm of my For and Rev primers = no PCR product?!? - (Feb/25/2010 )

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1.with that high of a GC content on the forward, i believe it is possible to achieve higher Tm value.
2. Instead of messing with the Tm value, have you tried using DMSO from 2-8%??
3. and try longer denaturation time.
Attached File


It worked! Thanks again to everybody. What the problem was - I still have no idea but
1. I used the suggested forward primer
2. DMSO and
3. ordered a clone with the cDNA I wanted to amplify and used it as a template

All together resulting in a very nice, sharp band in sufficient amounts.
Thanks people.

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