Ligation problems - (Jul/03/2005 )

Hi,

I have been trying to cut a 1.5 KB fragment out of pGEM T-Easy vector (3kb) and put it in another vector (5.0 kb). I cut my gene of interest from the T vector, gel purified it and did the same for the second vector. Then I set up a ligation reaction of the purified cut plasmid and the gene of interest and transformed them in competent cells. After doing restriction digestion of the plasmids isolated from these cells, I find that I am getting 2 bands of 1.5 kb and 3.0 kb, I am not able to see the band of 5.0 kb that is my new vector. Even after gel purifying my insert from the T-vector how am I getting the 3.0 kb band?

A line of reply will be highly appreciated. Thanks for the help in advance!!

Rishikesh.

Hi Rishikesh,

Any chance of sequencing the product. The only thing I can think of is that your initial digest was not efficient and there is some undigested pGem-T-insert in your band that was purified. This would result in the two bands 3kB (pGEM) and 1.5kB insert and also be transformed with much better effieciency than the ligated product. Did you run undigested next to the original digest could the supercoiled being running that low, a little odd but outside chance.

I think I would check a better restriction map with a number of enzymes or sequence the product. This should tell you what is going on. If it is pGem still then I would try to improve restriction digest efficiency and run gel longer for better seperation.

Cheers,

Scott

Thanks Scott. I picked up a few more colonies and got the expected results. I also found some information that hints contamination by the earlier vector. It is on this link http://www.protocol-online.org/archive/posts/6279.html Hope it helps people with similar problems.

Rishikesh.