Dear all,
In my next experiment i need to clone a large fragment(7kbp) of DNA into a (3kbp) backbone using Gibson assembly kit. NEB suggests using Q5 High-Fidelity DNA polymerase for this kind of cloning but i am not sure that the DNA polymerase will go through the whole fragment.
I am just wondering if anyone has a similar experience using Q5 High-Fidelity DNA polymerase, will it be ok to used it or should I look for another polymerase?
Thanks alot,
Expand Long Template PCR system kit from Roche works excellently. I learnt something very important from Paul Rutland some years ago, that the important thing in long DNA PCR is that the template does not re- anneal to itself before the extension happens. All long PCR should have primers that anneal at very high temperature to minimise the time that the template has to become double stranded again. Primers should be designed to anneal close to 72 deg C and do the PCR in the presence of 1M betaine which keeps the template melted for longer period by destroying the triple hydrogen bonding of G-C bonds which makes the template melt easier
Q5 is perfectly fine I have routinely used it for similar sized amplification. Also 7kb is not a large amplicon therefore it will work fine just use 30sec extension per kb.
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