can't make the cloning work - (Aug/10/2009 )

Hi
I am trying to ligate 650bp pcr product to PGEM-T and than heat shock transformation to E.coli competent cells.
it looks as if everything works fine, I got the colonies and I screen it with PCR, I get bands and send the same colony for sequencing. the results that I got back for all 6 replicates were "vector" in the BLAST serch.
what can be the reasons for that?
thanks a lot!
Einav

einav on Aug 10 2009, 07:12 AM said:

How are you digesting your vector? Single or double digest? If it's a double digest, how are you purifying the DNA? If it's by column purification rather than gel purification, check the size of the DNA you're cutting out of the vector with the digestion. It may be retained by the column and religating when you do the ligation.

If it's a single digest, then obviously it could also be religating.

Have you tried using a phosphatase on your vector?

einav on Aug 10 2009, 01:12 PM said:

What enzyme are you using for PCR? for pGEM-T cloning your PCR fragment needs to have A- overhangs at the end. If you are not using Taq, you have to add the A tail after purifying your product. Mix your PCR product with dATP, Taq and Taq buffer, and incubate at 70C for 15-30min. That should work, T cloning is usually pretty straight forward.

What primers are you using for the PCR screening? You might have only amplified vector, do you have a positive control for your PCR screen?

Didn't you transfer your colonies into master plate before colony PCR? This may be easy to be explained. I got this result sometime in the past, but now I can avoid that.
If you didn't change the plate, many PCR products (your gene) were still on the plate. And if you use the primers on the gene, you may got almost positive colonies.
Because sequencing is not that cheap and take time, I always check my samples very carefully before sending them. Do the digestion to confirm the presence of your gene, it's the best choice.
Hope that helps.