Few colonies frm sticky end cloning - (May/30/2013 )

Dear Scientists

I have been trying to clone a cdna insert 2.7 kb into pcdna3.(+) <5.4> kb via epicenter Fast-link ligations kit. After transforming my ligation I got serval clones. There was also significant amount of satellite colonies but I picked the colonies that seemed promising. My fragments have cohesive ends

Vector Preparation
Digested 5 ug of vector
After digestion CIP-ed the vector
Heat inactivated the BamHI and XbaI
Gel extracted vector 33ng/ul

The ligation was 3:1 molar ration insert: vector


From my picked colonies
I got a band that was approximately the size of my insert?!!!!

Any suggestions


Frustrated graduate student

So what's the problem? From your description you have done a ligation and got colonies that give you a band of the right size (which technique did you use here - PCR?, restriction digest?)

Satellite colonies indicate that your antibiotic selection has probably gone off - make up some fresh.

CIP can cause a lot of problems, with non-compatible sticky ends you shouldn't need to use it.

Sorry... I should get my backbone 5.4kb and insert 2.7. After running my digest all i am getting is 2.7 kb

I pcr-ed my insert with the respective sticky ends and did a ligation. I transformed into dh5-apha cells. AFter preping the colonies, the only visible band was around 2.7

Did you run some undigested and single cut plasmid along side as control? Are you sure you used the right plasmid in your initial cloning and if so, are you sure that the restrictions you have done result in single cuts (BamH1 and Xba1 are usually single cut in the MCS, but not always)?