Plasmid digestion - Problem with digestion (Aug/01/2005 )

Hi all
I have a problem with the digestion of the pCDNA Myc His(+) (A) vector.I am doing sequential digestions with Ecor1/Xho1 enzymes and running the controls with each step to confirm whether the enzyme has cut the plasmid or not and in the gel i am getting the desired results.
For tranformation i run the TEST along with the CONTROL
TEST:Vector + Insert
CONTROL: Vector only
Ideally if your vector is completely digested you should not get any colonies in the CONTROL
However, i get huge colonies in the CONTROL plate after transformation of the competent cells.
I have repeated the experiment three times but getting the similar results.

Kindly help me in troubleshooting this problem.
Rajat

P.S. The restriction enzymes i am using are from FERMENTAS and i have also looked at the possibilty that the ligase enzyme or buffer or Water might be contaminated. But i got negative results.They were ok.

and what about your test plate?
did you test colonies on your test plate?
i've seen huge results in the past and now i don't care any more about control. I do about 20-30 minipreps on the so called "test" plate and oi always get positives clones...

fred

Hi Fred
Well i have not checked the TEST plates but the the test plates did show huge colonies as well.
I have encountered this problem for the first time as previously my CONTROL plates used to give very few colonies as compared to the TEST.

That used to be a big assurance for me that my cloning was running fine.
Well ill check the TEST plates
Thanks for your reply

Rajat

Are you dephosphorlyating your vector or gel purifying your vector prior to ligation? You may be getting a huge amount of vector re-ligation if your "vector-only" plate has so many colonies.