Hi everyone,
I need to clone in pGEM-T vector the result of amplifying a metagenomic library by PCR with a high fidelity polymerase but I only want to clone the fragments of an specific size (1-4kb). The rest is not interesting. I was thinking about:
-Purifiying this PCR product
-Do a A-tailing
-Run an agarose gel to cut the band of the desired size
-Perform pGEM-T clonning
I am not sure if A-tailing will survive the whole gel process. Any idea or help with this?
Thank you so much beforehand.
Correct, you would need to remove high fidelity polymerase first.
No problem.
You could also change up the order of the steps.
Set up your PCR.
Run agarose gel & cut out desired band.
Gel purify band.
Do A-tailing.
pGEM-T cloning
Hi,
After your PCR reaction done just add 1 uL of 10 mM dATP and 1 uL of Taq. Thus, for A-tailing leave your reaction more 10 min at 72 0C. Next, you can purified your fragment from agarose gel and go to cloning in pGEM-T. Best wishes.
Yes, A-tailing will survive after electrophoresis. Purify the PCR product and perform the A-tailing reaction at 72 degree for 10 mins. Perform ligation with pGEMT vectore only after purification.
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