Mysterious plasmid preps

hardies at thorin.uthscsa.edu hardies at thorin.uthscsa.edu
Sat May 6 12:01:13 EST 1995


In article <3ob2l3$4eg at news.bu.edu>, rocket1 at bu.edu (Richard Near) writes:
> [Describes a typical 100 ml plasmid prep]...
> The peg precipitate is disolved in TE, and
> RNAsed for 1/2 hour and the subjected to 2 phenol-chloroforms. 
> Lastly it is ethanol precipitated and the redisolved in TE.
> 
> We find very high OD260 readings indicating a yield of 5 or more
> mg (a ridiculous yield for 100ml bugs). ...[Why?]

I'd guess that you RNAse is only partially digesting the RNA leaving
large enough fragments to co precipitate with the DNA, but small 
enough not to appear on your gel.  Try adding some Mg to the RNAse
incubation.  This is not that unusual a situation, and most people
just go on with it using the quantitation from the gel.  If you have
trouble restriction digesting the DNA, just add some more RNAse to 
the restriction rxn.  For DNA sequencing on this as template, expect
a solid grey background to your ladders.  It may be extensive enough
to require additional template purification.

Hope this helps.
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu






More information about the Methods mailing list