5 replies to this topic

[Anne G]

Hi all,
I have the following problem:
after transformation of XL-1 blue cells (form Quickchange kit, Agilent) with either pUC18 (control plasmid of the kit) or my pET plasmid+insert, the cells grow well on agar+Amp plates, but when I pick the colonies, inoculate them in freshly made LB+Amp medium and incubate them overnight @250rpm, 37°C, all I obtain the next morning is a cell lysate at the bottom of the culture tube (round bottom 14mL falcon). What could cause this unwanted lysis? Should I still attempt to purify DNA out of this lysate?
Thanks a lot for your help!
Cheers
A

[prabhubct]

Cell lysate you are considering may be overgrowth. Try to isolate plasmid.

[Anne G]

prabhubct, on 07 December 2012 - 05:31 AM, said:

Cell lysate you are considering may be overgrowth. Try to isolate plasmid.

Thanks for your answer!
Could be, but the rest of the incubating solution is clear... not the cloudy suspension you're supposed to observe

Edited by Anne G, 07 December 2012 - 06:09 AM.

[John Forsberg]

How long were the colonies on the selection plate?  Sometimes if the antibiotic is degraded near the colony, the colony can get rid of your plasmid and fail to grow.  That usually takes a substantial amount of time though.

Or maybe the plates are old?  Ampicillin is probably the least stable of the commonly used antibiotics in plates.  So maybe your colonies don't actually have your plasmid?

Another possibility is that your plasmid is expressing a toxic protein that is expressing enough in regular media to kill the cells after a little bit of growth.  Some media types (or commercial ingredients) can cause your cells to autoinduce after they reach a certain cell density.  Using catabolite repression for lac operon expression plasmids (adding 0.5% glucose or so) can prevent unwanted protein expression during your overnights.  If that might be a concern, then you can also look into Studier's autoinduction media papers/docs, for recipes that can suppress toxic protein expression.

[prabhubct]

Anne G, on 07 December 2012 - 06:09 AM, said:

prabhubct, on 07 December 2012 - 05:31 AM, said:

Cell lysate you are considering may be overgrowth. Try to isolate plasmid.

Thanks for your answer!
Could be, but the rest of the incubating solution is clear... not the cloudy suspension you're supposed to observe

Anne G, on 07 December 2012 - 06:09 AM, said:

prabhubct, on 07 December 2012 - 05:31 AM, said:

Cell lysate you are considering may be overgrowth. Try to isolate plasmid.

Could be, but the rest of the incubating solution is clear... not the cloudy suspension you're supposed to observe

tl

Culture should be cloudy overnight. But when we keep cultures in refrigerator often it settle to bottom, culture may be clean but bacteria present in them.
As John mentioned toxic protein expression may be reason. Or your cells have expressed desired protein in solution so much that it became toxic to your cells.

Hoping that you did not picked wrong colony you can isolate plasmid. meanwhile can inoculate same again in other LB+Amp will help you observe it again.

[Anne G]

Hi,
Thank you all for your answers. I isolated just a little bit of DNA from the lysed cells, enough to retransform into TG1 cells, which grow normally and express my DNA much better. I'll switch to this strain for the next mutagenesis.
Also tried to use minimal medium M9 with XL-1 blue cells, but obtained the same result: cell lysed already after overnight incubation. TG1 it is!
Good luck with your experiments!