cloning an AT rich gene - (Feb/06/2012 )

Hi,
I am new to cloning and facing a big problem.
I am trying to clone an AT (80%) rich gene from Plasmodium.
I have PCR amplified the target gene, extracted the band from the gel, purified it with Qiagen Gel extraction kit.

I run it on a gel to estimate the concentration and ligated it into PGem plasmid using the PGem Teasy kit in a 1:1 molar ratio (insert to vector).

Transformation looks o.k.
I did colony PCR with insert primers and got a strange size band, 1.2kb instead of 550bp? what could this be?

the other problem is when I try to sequence it with SP6 and T7 primers I only get the vector sequence.

did the ligation not work? how could I test it?


thanks

the ligation probably didn't work. that's why the vector sequence came up from sequencing.

how did the ligation plates look like? control vs ligation?

I don't like colony PCR. it gives false results. Contamination of unligated DNA from your ligation reaction might also be there when you spread your bacteria on plate.

If you are ligating the insert into the vector using restriction sites vs. blunt cloning, just do minipreps for some of the colonies and do a restriction digest to release the insert and then visualize that on a gel. I agree that colony PCR is very unreliable. I have observed the correct band from colony PCR for 10 colonies only to find out 8 of them were false positives.

Also, you could just linearize your ligation product using a RE with only 1 site and just observe the length