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Type of polymerases compatible for TA cloning

cloning TOPO plasmid

Best Answer phage434, 11 September 2013 - 12:43 PM

Taq polymerase or mixtures of Taq plus a proofreading polymerase will add a 3' A to the product, allowing ligation into the Topo vector. Pure proof reading enzymes such as Phusion, Q5, Pfu will leave blunt end products and will not clone.

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#1 pmggms2

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Posted 11 September 2013 - 11:39 AM

Good Afternoon All, 

 

I have a minor question about using the TOPO XL vector. I am trying to figure out what type of polymerase would you recommend to use for my PCR due to the 3' deoxythymidine (T) reside overhangs?

 

 

Thank you in advance  



#2 phage434

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Posted 11 September 2013 - 12:43 PM   Best Answer

Taq polymerase or mixtures of Taq plus a proofreading polymerase will add a 3' A to the product, allowing ligation into the Topo vector. Pure proof reading enzymes such as Phusion, Q5, Pfu will leave blunt end products and will not clone.



#3 bob1

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Posted 11 September 2013 - 03:30 PM

In addition, you can add an A overhang after the PCR by adding taq and dATP then incubating for 15 min at 72 C.



#4 pmggms2

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Posted 12 September 2013 - 07:24 AM

Thank you I really appreciate it 



#5 pmggms2

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Posted 17 September 2013 - 08:52 AM

So would you recommend hotstar high fidelity polymerase kit to be sufficient? 

 

#6 phage434

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Posted 17 September 2013 - 10:05 AM

If you are concerned about fidelity (which is perhaps not that important for 1000  bp fragments), then Bob1's answer would be best: do a PCR with a proofreading enzyme, then A tail with Taq and dATP. Otherwise, a Taq/Pfu mixture such as Invitrogen PCR Supermix would be an easy choice of enzyme. I like the master mixes, just because they avoid lots of pipetting and many tubes.



#7 pmggms2

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Posted 17 September 2013 - 10:49 AM

well the gene I am cloning is well over 1kb so I should be worried about fidelity especially since I will be using my product for sequencing 



#8 phage434

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Posted 17 September 2013 - 10:51 AM

If you are sequencing the PCR product directly, it makes no difference. If you are cloning and then sequencing, then it may be worth it.







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