digesting pQE30 vector gives inexplicable results - (Oct/18/2006 )
hi
i mistakenly (aka stupidly) digested a pQE30 vector (qiagen) with EcoRI and then realized (after 5 hours at 37C) that the vector does not contain a cut site for that enzyme. i then heat inactivated EcoRI and added BamHI (vector has Bam cut site) to the mix (i don't have any more DNA and couldn't set up a new reaction). i took aliquots of the mix with just EcoRI, after heat inactivation at 65C, and after treatment with BamHI.... and they ALL look the same. huh???? the pQE30 vector plus my insert both add up to about 4.4 kb, and the band on the EtBRr gel looks like it's just below the 5 kb marker-- which is only slightly comforting, but it shouldn't have been CUT BY THE ECORI!! (star cutting, perhaps?).
the original DNA was miniprepped from lab-made M15[pRep4] competent cells (which alone do not grow on Amp plates) containing the Amp-resistant pQE30 vector. one thing i found was that the untransformed lab-made competent cells do grow on plates containing both Amp/Kan-- but not on just Amp plates. i did a transformation and thought i'd plate them on Amp-only plates... two colonies grew on one plate (of 3 plates) from 100ul of the transformation reaction. there were no colonies on the control plates, which were just competent cells 'transformed' with water. when i plated the original source M15[pRep4] cells on Amp plates, they didn't grow; they grew on Amp/Kan plates. hence my reasoning for plating the transformation reaction on Amp only plates.
thanks for reading this loooong post. but i'm getting really tired of molecular biology these days. would appreciate any input--- very, very, very much. 
Sorry to say this to you, but pQE30 does have an EcoR1 site, just not in the His polylinker. The EcoR1 site is just behind the ribosomal binding sequence (RBS)
pQE maps
I would suggest that you midiprep more DNA and start over.
I find it rather strange... quite bizzare actually that you have untransformed cells that grow naturally on Amp/Kan plates but fail to grow on Amp only plates.
Amp and Kan work via different mechanism to kill the bacteria. I have not found any antagonistic behaviour between the two. I do know you can use double selection for Amp and Kan.
I must ask if your Amp/Kan plates are old or perhaps a mistake may have been made in their preparation. The antibiotics could have gone off.
pQE maps
I would suggest that you midiprep more DNA and start over.
I find it rather strange... quite bizzare actually that you have untransformed cells that grow naturally on Amp/Kan plates but fail to grow on Amp only plates.
Amp and Kan work via different mechanism to kill the bacteria. I have not found any antagonistic behaviour between the two. I do know you can use double selection for Amp and Kan.
I must ask if your Amp/Kan plates are old or perhaps a mistake may have been made in their preparation. The antibiotics could have gone off.
pQE vector series, up to me, is not a good choice. Although it have almost no leakage and some time the expression is excellent, I have several experience that no expression even the insert is successfully cloned into the vector.
Anyway, if you insist on using it, as perneseblue said, you should clarify the thing you are using but not just the agar plate.
(1) Digestion product is not the same as your expect
(2) Antibiotic resistance do not match your case
one possibility is that, the vector you using is not actually pQE30
If you cannot obtain more vector, try using PCR, and it need just tiny amount of your sample.
thanks for your help, folks! i didn't see the EcoRI site on the pQE as i was looking at the pQE30 MCS. i will be miniprepping more DNA as suggested, and i also am just going to do a before/after IPTG mini-culture experiment and run samples out on a coommassie gel, see if my protein is being overexpressed. if it is, i'll be sleeping better at night. if not, PCR cloning will become a very real possibility. thanks again.