Type of polymerases compatible for TA cloning - (Sep/11/2013 )
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
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.
In addition, you can add an A overhang after the PCR by adding taq and dATP then incubating for 15 min at 72 C.
Thank you I really appreciate it
So would you recommend hotstar high fidelity polymerase kit to be sufficient?
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.
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
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.