Vector construction - Can I use long PCR primers (60bp) and get good PCR poduct??? (May/11/2006 )

Hi everybody!!!

I am trying to clone flag tagged gene with gateway vector. I wonder can I do this with long primer like 60bp and get good PCR product?
My forward primer is getting very long 'cause i have to incorporate attB1 +flag (27)+(18 bp gene specific sequence) =60bp.
Or may be I have to first fuse the flag tag with my gene and than add attB1?

Is there any idea???

Than you in advance,
Lucised

PCR should work. I have done PCR with 72 bps primer and got good products.

lucised, the primary difficulty I foresee is that your reverse primer will have to be longer too because you might struggle to get equal Tm's

Hi!!!

Thank you very much!!!
I will use long primers and will prolong the reverse primer as well.
Best regards,

lucised

Practically anything will work with PCR, as long as it is carefully executed.

250+ bp megaprimers can work when properly purified (HPLC or PAGE).

-Matt