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does anyone have experience with pwo DNA polymerase for long PCR fragments ?


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#1 theofear

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Posted 28 January 2010 - 02:03 AM

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. :blink:

Edited by theofear, 28 January 2010 - 02:05 AM.


#2 chrisbelle

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Posted 28 January 2010 - 04:14 AM

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
Life sucks. Enjoy it while you can.

#3 theofear

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Posted 28 January 2010 - 07:07 AM

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.

Edited by theofear, 28 January 2010 - 07:09 AM.





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