I need to clone a 4kb gene. I have problem in cloning the whole gene in a single PCR reaction. So I decided to amplify two fragments then overlap the two fragments in a another round of PCR. Both of the two fragments are about 2kb long, having ~100 bp overlap.
I am not sure whether 100 bp overlap is too long for an overlap PCR? What are the specific conditions for the overlap PCR?
what is the annealing temperature should I set?
and how many cycles for the PCR without adding the outer primer pairs?
And is there a specific requirement for the DNA polymerase? right now I am using the Platinum Taq High fidelity DNA polymerase from Life technologies.
Thanks.
You can assembly partially complimentary fragments using nearly
any polymerase ( I use Phire II ), overlap could be less than 100 bp
(melting temperature should be above 72 ) but I don't see how 100bp would be a
problem. Less cycles will be needed than for "normal" PCR, I do 7 but you can play
with it. I would gel purify the fragments before assembly.
I have succesfully amplified overlapping fragments to construct an artificial microRNA. Basically I amplified the products separately (in my case 3) and then use them as DNA template in a fourth PCR with primers that match the 5' and 3' ends. For the last PCR I used 25 cycles and normal PCR conditions (94º 1 min; 55º 45 sec; 72º or 68º for amplification product-dependent). I also gel-purified the PCR products before the last PCR.
Good luck!!
Daniel
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