pET vector problems (HELP) - pEt 21 a (+) (Jun/06/2007 )

Hi,

At school, we use the pET 21a (+) expression vector system.
We have some problems with the transformation of our vector in E.coli BL21 (de3). All our samples and both positive and negative controls show the same amount of colonies (about 250). Our negative controls contain digested vector (dephosphorylated) without insert and only competent cells (no vector). Our positive control contain uncut pET vector. We use a 50 ug/ml concentration of Amp, which is recommended by Novagen.

Does anybody has some experience with this kind of problems?

-Roy van Heesbeen-

The vector is probably not completely digested and thats why you have the same no. of colonieson both the positive and negative plates. Verify if the enzyme is fine.

-scolix-

QUOTE (Roy van Heesbeen @ Jun 6 2007, 03:56 PM)

Our negative controls contain digested vector (dephosphorylated) without insert and only competent cells (no vector).

If competent cell itself still can grow on your plate same as your positive control, the plate is likely to be too old. This is common problem with Amp plates. Prepare fresh one, don't add Amp in when the agar is still too hot. The plates are likely not kept for long, only a week or so. Also, I personally prefer a higher conc than that, about 100ug/ml or above.

-Almasy-

I think that the amp. is the problem. My colleages probably poured the plates with a to high temperature.

We poured new plates, and try it again.

-Roy van Heesbeen-