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Can mutations be created during cloning?

cloning sequencing mutations

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7 replies to this topic

#1 Arrate

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Posted 12 September 2014 - 01:27 AM

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



#2 Rsm

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Posted 12 September 2014 - 03:37 AM

Which polymerase were you using? TA cloning with a proof reading polymerase can be a challenge, but it may improve reproducibility.


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#3 Raygoza

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Posted 12 September 2014 - 01:07 PM

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. 



#4 Arrate

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Posted 16 September 2014 - 03:14 AM

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?



#5 bob1

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Posted 16 September 2014 - 10:21 AM

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.



#6 phage434

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Posted 16 September 2014 - 01:37 PM

What Bob1 meant to say is Taq and some dATP (not ATP). Important difference.



#7 bob1

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Posted 16 September 2014 - 01:58 PM

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.



#8 Bio-Lad

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Posted 23 September 2014 - 07:21 AM

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).


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/






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