Electroelution/Centricon Problem

the End jgraham at bronze.ucs.indiana.edu
Thu Aug 31 16:50:12 EST 1995


Stop doing so much to your sample. DNA once electrophoresed 
in agarose becomes difficult to precipitate, and seems to be 
lost on conventional handling steps like tube transfers in my
experience (6 years daily).

Excize your low-melt gel slice (under LONG WAVE) UV, freeze it in a
microcentrifuge tube at -70 for 5 min, and spin briefly in the 
microfuge. Use the supernatant DIRECTLY in ligation, T7 transcription,
T4 endfilling, sequencing (EthBr OK). I have yet to see an enzymatic 
reaction which will not work efficiently on this substrate.

The major pitfall in isolation of DNA is to "overprocess" the 
sample beyond what is necessary for subsequent reactions.

Do you say "Kit"?  :)

Jim
J. Graham PhD 
Biology Department 
Washington University of St. Louis 

















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