2 replies to this topic

[JulieL]

Hi everyone,
I am doing 5'RACE using Invitrogen's Kit, then purifying my RACE product (I obtain a single band at 600 bp!) and then cloning it in TOPO-TA vector. Then I transform TOP10 competent cells, grow them on LB plates containing kanamycin, and the next day I screen the colonies (more than 30) by PCR using M13 Reverse and T7 primers and obtain various sizes of insert on the gel!!!! About 50% of the clones screened contain a truncated form of my gene (after sequencing) and I don't know where it comes from since I obtained a single band after RACE...

Does anybody already had that kind of problem???

Thanks for your help...

Julie

Edited by JulieL, 29 June 2009 - 09:42 AM.

[namekuji]

Hi Julie-  I wouldn't over-trust what you see in the gel. There are millions of unwanted molecules in the gel but you can't see them because they are below the detectable quantity. Nonetheless they can be preferred substrates for ligation (generally smaller fragments are easier to ligate).
You could try gel-purifying the 600 bp band before TOPO-cloning. That would at least enrich the DNA molecule you want.

[jiajia1987]

I agree with namekuji. I had this problem before when I simply purified my PCR products the normal way. Had a lot of different band sizes upon colony PCR and saw a lot of undesired sequences in my sequencing results.

Using gel purification will at least ensure that you get most of the product that you want to ligate to your TOPO vector. It would be good to clean your gel tank and use fresh buffer for gel electrophoresis in preparation for gel purification. Expose the gel tank to UV light for 5mins after washing it cleanly (you can soak the insides of the gel tank with water and virkon and wash it after that) to kill all old DNA molecules that may run into your gel and get purified along with your PCR products.