trouble with methlase sensitve restriction digests on plasmids isolated from DAM - (Mar/09/2006 )
Transformed my favorite plasmid (unmethylated- cuts with methylase sensitve restriction enzymes) into a DAM (-) E. coli strain (INV110-invitrogen) in order to isolate some more unmethylated plasmid (i was running low). After growing up the resulting transformants in the appropriate media and isolating the plasmids (average 150 ng/uL -miniprep), i went ahead and attempted a digest with a methylase sensitve restrction enzyme (ClaI). Much to my chagrin, the plasmid doesnt cut! This is surprising because i know this DNA contains a single unique Cla I site. The original plamid used to transform the DAM (-) E. coli cells cuts with this enzyme and was used as a positve control for all digests. I thought maybe i wasnt doing the microbiology properly, ie working with "unpure" poorly isolated colonies?...so i went through the trouble of isolating well defined colonies making sure not to over grow them....Isolated the plasmid and tried to cut with my methylase sensitive enzyme and again...the plasmid remains uncut...what am i doing wrong?...my next thought is that perhaps my restriction enzyme is being inhibited by something in the buffer that the DNA is solubilzed in.....The plasmid was isolated from cells using a miniprep protocol...the purified DNA is washed off small spin columns using an elution buffer...which is where the DNA remains...not sure what the components of this elution buffer are ...i think this one is a long shot though....or maybe my cells have gone south?!..not doing what they are suppossed to be doing?...what am doing wrong here?...anyone out there know?...id love some suggestions if you guys have em! THANX!
Have you used "freshly"cultured coli's, the fresher there are from the glycerol stock, the better.
And are you sure your ClaI is still working?
I really don't know, it should work. Only thing I can come up with is that either the coli's are not a DAM- strain or that your enzyme is not working anymore.
I've never had problems with miniprep colums.
Have you sequenced the new isolated plasmid? Maybe there is a mutation. Just to be sure if the ClaI site is still there.
And are you sure your ClaI is still working?
I really don't know, it should work. Only thing I can come up with is that either the coli's are not a DAM- strain or that your enzyme is not working anymore.
I've never had problems with miniprep colums.
Have you sequenced the new isolated plasmid? Maybe there is a mutation. Just to be sure if the ClaI site is still there.
hey aspergillie...thanx a bunch for your response! As far as i can tell the cells (INV110) are "fresh"(i think), as they were purchased from invitrogen (a few months ago) and stored @ -70 C. Maybe purchasing some new cell aliquots will alleviate this trouble. I know my enzymes are working because my positive control still cuts (from the same aliquot of Cla I enzyme). I hear that DAM- strains tend to have a higher mutation rate, sure wish that sequencing was an option. Could it be possible that i didnt do the plamid isolation cleanly and that something (some nasty salt or something) from that protocol was carried over into my final product?...something that would inhibit my enzymes activity? I think im gonna try to dialyze my plasmid samples, dry them off, and reconstitute them in some fresh buffer and attempt to the digest again just to convince myself that something must be wrong with my DNA or the coli strain im playing with....i hope it works...if not maybe its time to call invitrogen for some new DAM(-) cells....what do ya think?...thanx again!