use PCR for cloning of long fragment??
simonssp at pfizer.com
Fri Oct 11 06:04:58 EST 1996
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!
The preceding is a non payed for non advertising announcement. Any
resemblence to persons living or dead is purely coincidental. These
views do not necessarily represent those of Pfizer, Inc. nor are they
intended as an endorsement for any commercial product mentioned. Any
lawyers listening in should bugger off. Thank You.
More information about the Methods