how to get rid of a plasmid? - (Sep/16/2008 )
Hi!
I need to get rid of an Amp positive low copy plasmid in my bacteria. There is a BAC clone in them with kanamycine resistance. I thought to culture them in Kanamycine, with serial dilutions. Does anyone have an idea, how long should I grow them without Amp in order for the plasmid to be gone?
Thanks!
R
I need to get rid of an Amp positive low copy plasmid in my bacteria. There is a BAC clone in them with kanamycine resistance. I thought to culture them in Kanamycine, with serial dilutions. Does anyone have an idea, how long should I grow them without Amp in order for the plasmid to be gone?
Thanks!
R
When curing my cells of a plasmid, I grew them for three days, diluting the cell culture with non-selective medium twice a day. On the third day, I plate some of the the cell out to get single colonies. The next day I tested about 100 colonies (by streaking onto non-selective plate and selective plates) for the lost of plasmid.
Thanks!
Was it actually necessary to screen 100 colonies? About how many lacked the plasmid?
um no. If I remember correctly about 40% of the colonies had lost the plasmid. So it wasn't necessary to pick 100 colonies.
I have also heard (though never tried) that you can cure a strain of a low-copy number plasmid by electroporation. Seems that the electric-field induced membrane changes that can allow plasmids in can also let them out...
There are other harsher methods (acridine orange, for example) but these types of mutagens may also cause a mutation in the chromosome.
Naiive question, but why can't you purify the BAC from your bacterium and transform another cell that doesn't have the amp plasmid?
In term of low copy yea. keep doing the serial dilution you will get it. as long as it's low copy i m talking less than 7 plasmid per chromosome. that would work.
some plasmid due to their high copy number is nearly impossible to get rid of /cure.
Yea you could try what swanny is suggesting. isolate the plasmid out and pop it back .
It's possible . u just need to know the size of both plasmids so that you know which one to cut out. hopefully that helps.
In term of low copy yea. keep doing the serial dilution you will get it. as long as it's low copy i m talking less than 7 plasmid per chromosome. that would work.
some plasmid due to their high copy number is nearly impossible to get rid of /cure.
Yea you could try what swanny is suggesting. isolate the plasmid out and pop it back .
It's possible . u just need to know the size of both plasmids so that you know which one to cut out. hopefully that helps.
You could also try transforming with another incompatible plasmid (same ori) with a different marker. For example, same ori (pBRori) with ampR you can cure by transforming another plasmid with pBRori with kanR and grow in 30 ug/ml kan. They usually do this for higher copy plasmids.
Hope that helps.