extremely low copy number plasmid
tfitzwater at somalogic.com
Thu Jan 31 10:32:10 EST 2002
>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.
>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
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