PCR primer problem ?

redeamer alion85 at hotmail.com
Wed Jan 21 01:11:22 EST 2004


Hi!

1) Are you absolutely sure that this primer have worked before?

2.1) if no then what is the target of amplification? if it resides in
genomic DNA then you will really have problems with its amplification
even with a 21 bp primer if it is a single-copy gene for example. If
it resides in plasmid 15-20 of primer-template identity should be
enough...

2.2) if yes... then if every component you had in your mixture with a
working primer remained constant then you have to book a new
adaptor... you ll loose time..but you could try different
PCR-enhancing components such as DMSO 1-10%, gradient PCR for
annealing, i had an experience of actual annealing temperature 15
degrees less than predicted (Tm - 5) for a particular primer :)

3) hardly anything coould happen to your primers unless you spilled
some Dnase there

Cheers,

Drew



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