does anyone have experience with pwo DNA polymerase for long PCR fragments ? - (Jan/28/2010 )

I'm trying to amplify 4.5Kb from cDNA with pwo DNA polymerase - and without results (with gradient of Temp. ) ...
Do I need to change my enzyme ?

thanks.

Hi,

Are you sure your cDNA is 4.5kbp? How did you know that? Did you BLAST it or something? What primers did you use for your reverse transcription and what organism did you extract the RNA from? Are you sure your RNA is OK?

Chris

Hi Chris

I already have a cDNA, and I want to amplify from my cDNA only this fragment of a gene (4.5 kb) - actually it's the 3' UTR of the mRNA.
I have for this fragment 4 primers :

3'-----fragment1(600bp)-----------------------fragmen2(2200bp)----5'

I get the product of these two fragments (1 and 2) , but now I want the two fragments and what is in between (about 4.5 Kb),
I use the forward primer of fragment1 , and the revers primer of fragment 2, BUT I get no product , so I think
that my problem is with my anzyme.