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What are the error-rates of the TA cloning Kits?


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#1 Arrate

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Posted 17 September 2014 - 04:02 AM

Hello,

 

Does anybody know what is the error-rate of the TA cloning Kits of Invitrogen?

I mean the errors that can be created in the cloning process, and after sequencing the different clones, you see as variations in the DNA sequence (but are errors).

 

Thank you



#2 Bio-Lad

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Posted 23 September 2014 - 07:17 AM

I'm not aware of any significant error rate in the TA cloning step itself.  If anything the errors would be more likely to have occurred during the PCR amplification step. What polymerase are you using and what sort of sequence are you amplifying (are there repeats, etc...)?


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#3 Arrate

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Posted 23 September 2014 - 11:34 PM

I'm not aware of any significant error rate in the TA cloning step itself.  If anything the errors would be more likely to have occurred during the PCR amplification step. What polymerase are you using and what sort of sequence are you amplifying (are there repeats, etc...)?

 Hello,

 

I am using the BIOTaq DNA polimerase. I know that there are more faithful polymerases, but I need one that leaves an adenine overhang, in order to do TA cloning.

And the sequence which I want to analyze has a copy number polymorphism, I want to sequence the different copies, independently.

 

Thank you,



#4 Bio-Lad

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Posted 24 September 2014 - 03:31 AM

Well there are blunt TOPO coming kits available or you can add the overhangs yourself after PCR but especially if you have repeats and such, I'd highly recommend a more high fidelity polymerase. I tend to use Phusion from NEB.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#5 Arrate

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Posted 24 September 2014 - 11:30 PM

Well there are blunt TOPO coming kits available or you can add the overhangs yourself after PCR but especially if you have repeats and such, I'd highly recommend a more high fidelity polymerase. I tend to use Phusion from NEB.

 Thank you very much for your help. I will use a more high fidelity polymerase and use kits for blunt ends or add overhangs and use TA cloning kits. I don´t know which is the best option.



#6 Bio-Lad

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Posted 25 September 2014 - 02:21 AM

Which is best depend on your preference. If you already have a TA-TOPO kit, then you may not want to spend the $300 our do to get a blunt cloning kit. In that case, column purify your PCR product and add the overhang.

If you don't want to add overhangs (it's a simple step, really) and don't care about the cost, then purchasing a blunt cloning kit or dying if someone your building will give you a single reason's with of reagents may the best option.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#7 Arrate

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Posted 25 September 2014 - 05:15 AM

Which is best depend on your preference. If you already have a TA-TOPO kit, then you may not want to spend the $300 our do to get a blunt cloning kit. In that case, column purify your PCR product and add the overhang.

If you don't want to add overhangs (it's a simple step, really) and don't care about the cost, then purchasing a blunt cloning kit or dying if someone your building will give you a single reason's with of reagents may the best option.

 

I have TA-TOPO kit, so I want to use it, but using a high fidelity polymerase for the PCR. I was concerned about making more errors in the process of adding overhangs, because Taq polymerase has to be used. I have been told that to add overhangs I can incubate muy PCR product with Taq polymerase, and dNTPs (only Adenine), at  72ºC.

Is it a good method?



#8 Bio-Lad

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Posted 25 September 2014 - 09:58 AM

Yep, that works.  I wouldn't be worried about the errors from Taq in this step.  Taq's errors are made when extending the sequence you're amplifying but you aren't actually doing any amplification in this step.  You just incubate for the A overhangs.  It shouldn't affect the fragment you've already amplified using the high fidelity polymerase.  You will probably want to either gel purify or column purify your product before adding the overhangs to get rid of the other polymerase, which may chew those ends right back off and give a low number of clones.


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#9 Arrate

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Posted Yesterday, 03:01 AM

Yep, that works.  I wouldn't be worried about the errors from Taq in this step.  Taq's errors are made when extending the sequence you're amplifying but you aren't actually doing any amplification in this step.  You just incubate for the A overhangs.  It shouldn't affect the fragment you've already amplified using the high fidelity polymerase.  You will probably want to either gel purify or column purify your product before adding the overhangs to get rid of the other polymerase, which may chew those ends right back off and give a low number of clones.

 I will try to add the A overhangs, after purification with colimns.

Thank you very much for your help.






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