I have an E. coli BL21(DE3) strain which contains two plasmids (pCDF and pACYC). I need to isolate pACYC alone from this base strain. Could somebody suggest me an easy method?
FYI, I already try to isolate plasmid by mini prep. I got smear rather than two distinct bands. It could be due to the tertiary structures.
It's normal that you get smear, because the plasid are not linerised.
The two plasmids should have different antibiotic resistance. For example if pCDF is AmpR and pACYC is CamR you can culture the strains in media with ampicilin or cloramphenicol depending on the plasmid you want to keep or lose. After a few generations the cells should have lost the plasmid they don't use, as bacteria usually don't replicate what they don't need.
Another way would be doing the miniprep you are doing, and later using an endonuclease that cuts both plasmids in only one position each, so you get two linearised constructs. You then run the digestion in a gel and you can purify the linearlised plasmid of interest from the gel, ligate it again and there you have it.
Best luck!
You might need several steps to get rid of pCDF plasmid. Without antibiotic selection, some plasmid can be lost during the bacterium growth. What you need to do is grow liquid over night culture of your E. coli BL21(DE3) with chloramphenicol, after that streak some of your o/n culture onto a L-agar plate containing 25ng/ml chloramphenicol and incubate over night to get some single colonies (don't streak too much culture, otherwise you will get colony streak rather than single colonies). Label each single colony and than steak a few of them onto a L-agar plate containing ampicillin respectively, after over incubation at 37"C, these not growing in the Amp late indicate that they get rid of the pCDF plasmid. Go back to your Cam plate and grow one of these (not growing in Amp plate) in LB medium containing chloramphenicol for your plasmid preparation.
Simply take one aliquot of your miniprep, dilute it 1/100 and use it to transform a decent endA- strain, plating on a medium that selects for the pACYC plasmid. The chances of both plasmids co-transforming the same cell are much lower than those of a single plasmid.
Would you get the result yet ? Please let me update here how you will get success. Miniprep or antibiotic selection ? Thank you !
Thank you all for your valuable suggestions. Currently I am working on both antibiotic selection and miniprep digestion using enzymes. Hope I will get single plasmid through any one of these two methods.
All these methods are sounds good, please update here after finishing your experiments, which one method you got better?
Hello friends, I could cure one plasmid by transferring continuously to the medium containing single antibiotic. I did nearly 12 transfers to get rid off the other plasmid. Thank you very much for all your support.
I tried digestion of miniprep plasmid (mix) by endonuclease which cuts both the plasmids at single position. It did not work well. It still showed a smear while running in gel after digestion.
Hi BL21 is a rich endA+, So, after miniprep it digests your DNA and making smear on the gel. Some miniprep kits provide two washing buffers, one is to eluminte endA+. For example QIAprep kit from GIAGEN have two buffers one called PB buffer which is required when using endA+ strain like BL21. Hope this help Regards
- Badges
- Science topic