Plasmid lost during culture - (Jun/17/2008 )
Hey all,
I've been trying to get a miniprep of some clones recently but the plasmid seems to be disappearing in the culture. Half the clones grow and half do not but even the clones that do grow have no plasmid after the miniprep and digest. It's a low copy vector so i'm culturing in 50 ug/mL ampicillin. I think the plasmid is either being lost by not being selected for or the plasmid is being integrated into the genome. How likely are either or these options? How can i prevent the plasmid from being lost?
Cheers, Rob
What culture volume are you using? I use 3ml for my low copy plasmid for a mini prep. Do you have loads of satellite colonies?
5 mL culture volume. Not many satellites. I use 150 ug/mL ampicillin plates to avoid satellites but culture in 50 ug/mL ampicillin to not add to much pressure.
Perhaps you should repeat your transformation and start over
Perhaps you should repeat your transformation and start over
Already done that! I diluted the template to get fewer colonies (which i did - about 100 or so) but still have the same problem of no plasmid after miniprep. Thanks for the interest.
Are you sure your Amp is ok? When you did the transformation did you do a control plate? ie: Just plate the cells without DNA?
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Already done that! I diluted the template to get fewer colonies (which i did - about 100 or so) but still have the same problem of no plasmid after miniprep. Thanks for the interest.
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Which strand of bacteria are you using?
cause i had a similar problem some time ago using BL21 bacteria which are suited for protein expression and not for subcloning.
Try to do the mini-prep and run that on a gel without digesting and see if you get the right bands.
Some strains, like BL21, have high level of DNAse which elute with the plasmid when doing the mini-preps so when you digest it you'll destroy all the DNA and that may be why you don't see anything.
How do you do your miniprep? Is it with homemade buffers or kit?
I had the same problem (it's even posted somewhere in the forum). None of the mini-, midi-. maxi kits has worked. Try to do the miniprep with alkaline lysis method (homemade buffers).
The amp is ok because i had a transformation with the same cells the other day that gave me no colonies.
cause i had a similar problem some time ago using BL21 bacteria which are suited for protein expression and not for subcloning.
Try to do the mini-prep and run that on a gel without digesting and see if you get the right bands.
Some strains, like BL21, have high level of DNAse which elute with the plasmid when doing the mini-preps so when you digest it you'll destroy all the DNA and that may be why you don't see anything.
I use XL10-Gold from Stratagene. Yes, the reason i know there is no DNA is because the digest has been giving me no bands - just a blank gel. I've used this strain for a long time now and find it is generally pretty good. The problem of no DNA seems to occur with this vector though, that seems to be the common factor.