| S.Sandhya said: |
| Sequence is cloned to p Bluescript vector and transformed to E.coli. Selected cloned aquired ampicillin resistant , but plamid is not visible in agarose gel. I wish to know what could be the reason? How to increase copies of plasmid? |
Following transformation, did you plate out the bacteria on LB-amp or just pour the transformation mixture in to LB broth? If you plated out - did you get well defined colonies on the plate or a lawn?
SD's point with forgetting the amp in the overnight culture sounds likely (who hasn't done that once....or twice?)
I had a similar problem once when I cloned the human elafin cDNA into pBluescript (and pUC) to do SDM on it. The problem was (i think) that elafin is known to have a bacteriocidal effect and thus there was heavy selection for clones that lacked the elafin and yes, there were clones that didn't even have the plasmid backbone either. Could your sequence be toxic?
I got round the problem by cloning the elafin cDNA into a pBluescript that already had coding regions on each side of the insertion RE site. This prevented expression of elafin from the T7 and T3 bacterial promoters.
There is one other possibility - but I'm sure it's not likely: EtBr in the gel?