use PCR for cloning of long fragment??

sam simons simonssp at pfizer.com
Fri Oct 11 06:04:58 EST 1996


Guido,

We routinely use Boehringer's Expand PCR for long templates. It also 
improves fidelity over Taq as it is a mix with Pwo. I'm not sure how 
well Pfu works on long templates but it has much better fidelity. Just 
remember when working with proofreading enzymes that they extend an 
order of magnitude slower than Taq so your extension times must be 
increased to about 2-3 minutes per Kb (10 min for 4 Kb).
For cloning in baculovirus it is important not to have too much 5' 
untranslated sequence, especially if there are ATGs present (in any 
reading frame). Eukaryotes pick the first ATG they see. It is also 
important to have a kozak sequence immediately in front of your 
initiating ATG. I would suggest elimination of the 5' untranslated 
sequences during the PCR. Good luck!

Sam

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