No plasmid in HB101 - whereas DH5alpha does (Feb/02/2006 )
I did transformations of a ligation (7kb + 8kb, I know it's a big one), in both DH5alpha (chemically competent) and HB101 (electro competent) cells. Plated them out and picked from both plates about 12 colonies. Digested the preps and ran the gel: No plasmids! visible from the HB101 colonies, these lanes were completely blank, no smears, no DNA sticking in the slots whatsoever, whereas the DH5alpha colonies gave nice (although wrong) and clean plasmids.
I did the DH5alpha and HB101 transformations on the same day, same SOC, same LB-Amp, same miniprep solutions, shaking together, everything went parallel. The cultures grew and I thought I had a nice DNA pellet when I finally dissolved it...
Any idea's about what's going on? When the preps turn out to not contain the desired ligated vector (as this was the case for the preps from the DH5alphas), I can live with that. But no plasmid at all is much more frustrating! (I hoped the electro competent HB101's would have a better chance to accept my relatively big ligation).
HB101 is not known to discard plasmids easier than DH5alpha, is it? Should I try carbenicillin or growing in Super-/Terrific Broth?
Kind Regards, Groetjes, Grüße,
HB101 is endA+, unlike DH5a. This means that nucleases can degrade your plasmids easily. You need to do the extra washes of your plasmid DNA and work quickly to prevent DNA degradation.
Phage434, thanks for your reply! But wouldn't you expect to see at least some plasmid or their degradation products?