One-Step Miniprep for Screening by Size

From BioTechniques24:748-750 (May 1998)

Inoculate colonies into 3 ml LB+Amp, grow ON at 37°ree;.
Take 150µl of saturated culture, spin down cells, discard supernatant.
Add to cells: 40 µl Sucrose Loading Dye (0.1% BPBlue, 6% Sucrose) and 14 µl of 1:1 phenol:chloroform.
Vortex 5-10 sec to (mix and) lyse the cells.
Spin 3 min. to separate aqueous from organic phase. Cell debris will form a barrier between the 2 phases.
Remove 10µl of aqueous (upper) phase and load directly onto agarose gel (1-1.2%), with a sample of the original vector as a reference (Run gel).
Stain gel with EthBr to view the nucleic acids.
(From these samples, pick only the interesting prospects to do a real miniprep on).

FOR SAME-DAY DETERMINATION: Instead of growing cells overnight ...
Pick colony and grow up in ~0.5 ml for 5-6 hours. (LB + Amp, shaking, 37°ree;C).
Use the whole culture in the miniprep procedure above, except leave enough to inoculate an overnight culture for a real miniprep the next day.

Shirley Reynolds

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