Mysterious plasmid preps

Robert Hutton hutton_rl at delphi.com
Thu May 11 13:34:50 EST 1995


<hardies at thorin.uthscsa.edu> writes:
 
>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've found that even the slightest amount of phenol left in the mix will absorb
HUGE at 260 nm (actually, it shifts the peak over to about 264, I believe). 
This can be cured with a choloroform only extraction afterwards.  Another way
to check your concent
ration is a plate filled with 0.6% agarose and 1 ug/ml ethidium bromide.  Use
1-5 ul of your DNA and compare to varying concentarations of some known
standard you have hanging around.  This is much more reliable than the 260
absorbance method as long as you trust what your eyes tell you.
 
SGT Robert Hutton
USAMRICD
hutton_rl at delphi.com



More information about the Methods mailing list