overlapping PCR protocol - (Feb/09/2012 )
pDNA on Sun Feb 19 19:11:52 2012 said:
Phusion seems to be the only polymerase that works fine for overlap extension PCR (according to the paper referenced above) ...never used a different enzyme ...and if the paper is right ...i would not recommend to do so!
No, i never use phusion for overlapping pcr but i still got nice band,
as i told you using combination of polymerase of pfu and taq could help (for second pcr)
In addition to that, most of people recommend to pcr first fragment with pfu or vent polymerase
this is because the polymerase used here will not add poly A...
let say your gene: NNNNNNNNXXXXXXXXX + XXXXXXXXXXXJJJJJJJJJJJJJJJJJ
if you use some polymerase that produce A tails the first fragment will be AAAANNNNNNNNNXXXXXXXXXAAA
the A will interfere from bind to XXXXXXXXJJJJJJJJJJJJ but that's not critical....sometimes if you are lucky you still can have fusion pcr very well...but the chance is..................
using a mix of polymerases is what they do for the Stratagene Quikchange kit ...where u have "short primers" (~30-60 nt) overlapping primers ...the method i referring to uses "megaprimers" ...so a whole fragment (>100 nt>) as a primer ...and it seems that here Phusion is the only polymerase that is able to yield sufficient amounts of colonies ...so we shouldn't mix protocols here and not confuse people.