extremely low copy number plasmid

[Trond Erik Vee Aune]

Alison Leakey wrote:

> Hello everyone!
> 
> I would appreciate any suggestions. I am working with a plasmid that is
> in extremely low copy number. Lately we have been trying to increase the
> yeild of our preps. Normal mini-preps give us nothing - no visible
> plasmd. The QIAGEN maxi prep kit with cultures of up to 2L gives us very
> faint bands that are relatively resistant to digestion.  Alkaline lysis
> yeilds similar faint bands but also poor quality DNA. We have also
> increased the concentration of selective antibiotics in cultures with
> only marginal success. We are just about to give up.
> 
> Any thoughts??


I work with a plasmid with a copy number of 4-5. I have no problem 
isolating plasmid from this with standard mini-prep protocols and still 
get reasonable yield for cloning. Whenever I do this I reduce the final 
volume to 50 microliters (instead of the normal 100 microliters) and I 
also cut 6 microliter of my prep to get fat bands on the gel.

Of course your copy number may be even lower than mine, down to just 1 
per cell. In this case you'd have to cut 4-5 times more than I do, or 
simply concentrate your prep after isolation. You might even want to try 
to reduce the final volume even more, down to 30 microliters.

There may be other reasons for your problem, though. What kind of strain 
do you use (some strains are not good for isolation of plasmids)? How 
large are your plasmid? What type of plasmid is it? What type of 
selection marker do you use?

Kind regards,
Trond Erik Vee Aune

http://kibt.chembio.ntnu.no/molgen/