Can mutations be created during cloning? - (Sep/12/2014 )
Hello,
I have cloned my PCR product using the TOPO-TA cloning kit (Life Technologies) and the pCR2.1-TOPO vector.
When I sequenced the different clones, I saw that there were many differences between them and with the sequence of reference. Nearly in each clone appeared one different mutation. I expected to find some differences, because the gene shows CNV polimorphisms, but the variability observed seem to be too high.
Can some of these mutations have been created in the cloning process?
Has anybody had problems with this kit?
Thank you
Which polymerase were you using? TA cloning with a proof reading polymerase can be a challenge, but it may improve reproducibility.
Mutations can arise during ligation, restriction, even during while they're being replicated in the bacteria, this is more likely to happen if it is a high copy vector.
Rsm on Fri Sep 12 11:37:34 2014 said:
Which polymerase were you using? TA cloning with a proof reading polymerase can be a challenge, but it may improve reproducibility.
Hello,
I am using BIOTAQTM DNA Polymerase. It has 5´-3´ exonuclease activity, but doesn´t have 3'->5' exonuclease activity.
Which polymerase should I use to reduce the error-rate?
Any proofreader should work. To get the A overhang all you need to do is purify the PCR product then incubate with Taq and some ATP (and buffer) for 15 min at 72.
What Bob1 meant to say is Taq and some dATP (not ATP). Important difference.
phage434 on Tue Sep 16 21:37:07 2014 said:
What Bob1 meant to say is Taq and some dATP (not ATP). Important difference.
Whoops, yeah... dATP, my only excuse is that it was very early in the morning when I wrote that.
You can use a high fidelity polymerase like Phusion or Q5 and if you don't want to add the overhangs after the fact, do the TOPO cloning to a blunt vector (Invitrogen has their pCRBlunt-TOPO kits).