ccdB survival E. coli transformation - (Dec/30/2009 )
We are working with pWATERGATE plasmid (18050 bp). We have transformed this plasmid into ccdB survival E. coli (Invitrogen) by using heat-shock method, and then spread it on LB plate containing Spectinomycin 10 microgram/ml and incubated overnight, then subcultured the colony into LB broth containing 10 microgram/ml Spectinomycin, then isolated the plasmid. We isolated the plasmid by using Alkaline Lysis with SDS method (Sambrook and Russell).
In order to confirm this plasmid, we did double digestion (according to the restriction map, this plasmid has one restriction site for SacI and XhoI, so after digestion it would produce 2 band; the size is about 16.3 kb and 1.7 kb).
In our case, after digestion we only got one band (the size is about 9.5 kb).
Does it mean that we have isolated incorrect plasmid?
Is it possible for untransformed ccdB survival E. coli to grow on media containing antibiotic (10 microgram/ml Spectinomycin)?
Thank you in advance.
strange?! ...seems like the wrong plasmid? But if you use purchased cells they should contain no other plasmids!
Maybe your plasmid gets degraded or some sequences are deleted after transformation.
Do you did negative controls? Transform your cells with blank water? Maybe its a contamination!
Yes, we did it, and we could not get any colony from this negative control.