Toxic Plasmid?? Toxic Insert??
Posted 29 March 2009 - 09:27 PM
However, when growing these clones in DH5alpha cells, Glyt2a grows great - large bacterial pellets and corresponding high yields of plasmid DNA, whereas Glyt1b is total crap! Low growth and low yields. I suspected Glyt1b may have picked up a mutation in the F1 origin, so I cloned the insert back into the vector that had previously housed 2a with the same results - crap growth and yield. I typically recover only 4 ug of 1b from 10 mL of LBA culture (and before you blast my minis, I get 15-20 ug from 5 mL of 2a done side by side).
I have always heard rumors about DNA inserts that are toxic to host cells, but I have never encountered one - and certainly not one that would be toxic in an environment that is not actively promoting protein expression in the bacteria. Does anyone have any experience with anything like this? I've tried growing in TB and growing for 24 hours, with no increase in culture turbidity. Would it grow better in another strain? Different temperature? Different vector? Is T7 'leaky' in DH5alpha cells? Could one glycine transporter be more toxic than a closely related family member? HOW??? Is there a resource for this besides word of mouth and tall tales told over cocktails?
Captain, I've got to have more DNA!
Posted 30 March 2009 - 12:37 AM
Posted 30 March 2009 - 04:31 AM