My TOPO-TA cloning did not work. - (Jul/11/2008 )
QUOTE (higginsb @ Jul 14 2008, 11:02 AM)
QUOTE (dc11 @ Jul 11 2008, 01:05 PM)
Hello all,
I am trying to clone my PCR product (2.6kb) into TOPO-TA vector. I got a single, strong, and discrete band from PCR. I was using high-fidelity Taq. I used 3 ul of the PCR product for the cloning and incubated for 30 min. But there was no any colony in the plates. The TOPO-TA kit is fresh. Can anybody give me your kind suggestions?
Another question, does dNTP mix rather than dATP work for addition of 3' A-overhangs after gel purification of the PCR product?
Thanks a lot in advance for your help!
DC
I am trying to clone my PCR product (2.6kb) into TOPO-TA vector. I got a single, strong, and discrete band from PCR. I was using high-fidelity Taq. I used 3 ul of the PCR product for the cloning and incubated for 30 min. But there was no any colony in the plates. The TOPO-TA kit is fresh. Can anybody give me your kind suggestions?
Another question, does dNTP mix rather than dATP work for addition of 3' A-overhangs after gel purification of the PCR product?
Thanks a lot in advance for your help!
DC
Just a shot in the dark, but have you run out a microliter or two of the gel-purified DNA to make sure it is there? Sometimes the kits fail. It's unlikely, I know, but it's a control I always run now just to make sure. Just out of curiosity, why did you cut the fragment out at all? Why not clone directly?
Good luck.
Hi, I just did the cloning again after I purified the PCR product, and this time it worked out and there were a lot of colonies. Last time I cloned directly using PCR product but it did not work. Thanks.
-dc11-