Can someone explain if there is any importance in having nick (e.g with Taq polymerase) or blunt (Phusion TM) ends while running QuickChange?
I read something like> "As the newly synthesized DNA is nicked, it can not be used as a template for subsequent amplification" (and then high amounts of template should be used?) If I use Phusion or Pwo polymerases, which produce blunt ends, is this problem avoided?
I also read that the product is repaired by an E. coli host system. Does it happens with both, blunt or nick ends?
The problem with the Taq is not an issue with nicking. The issue is that Taq adds Adenosines to the 3'-end of the PCR product (which is also the reason that PCR reactions for TA cloning should always be done with Taq).
Phusion polymerase generates a blunt-ended PCR product (linear), which circulizes into a nicked plasmid. The nicked plasmid is repaired by the bacteria, thereby generating the desired circular plasmid.
The low cycle number is to avoid oligomerization i.e. repeated primer sequences inserted at the nicked site.
I hope this clarified things.
I do not know what you mean by nick with Taq. Taq adds an A onto the 3'end of the amplified product. There is no nick. I would suggest you use Pfu Turbo, Phusion or other similarly high fidelity polyermases. The reason to not use Taq is because it is lower fidelity and really struggles to be accurate the longer your PCR product. This is partially due to dUTP build up. You can add a dUTPase to improve this (Turbo fragment of Pfu Turbo, for example). Transformation of your PCR product with two nicks should be repaired by the bacteria. We abandoned this PCR site directed mutagenesis protocol due to the high number of unmutated colonies we were getting and used the NEB protocol developed for Phusion. However, we substituted PFu Turbo and incorproated the DpnI step from Quickchange and now our success rates on first attempt is >92%.
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