extremely low copy number plasmid

[Tim Fitzwater]

>Subject: extremely low copy number plasmid
>From: Alison Leakey (leakey at cber.fda.gov)
>Date: Wed 30 Jan 2002 - 21:43:03 GMT 
>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 
>yield of our preps. Normal mini-preps give us nothing - no visible 
>plasmid. The QIAGEN maxi prep kit with cultures of up to 2L gives us very 
>faint bands that are relatively resistant to digestion. Alkaline lysis 
>yields 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?? 
>Alison Leakey 
>PS. We have used these methods with other low copy plasmids also with 
>good results so we are not making any obvious mistakes. 

If the plasmid is ampicillin resistant, you may be able to select for a high
copy number version by plating cells on plates containing methicillin.  
Otherwise, try chloramphenicol amplification (as described in Molecular
Cloning).  

Tim Fitzwater
SomaLogic, Inc.

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