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PLASMID MINI PREP Inoculate 5 ml YT + Amp with single bacterial colony. Incubate at 37 C over night with
 

Summary: Auble Lab
PLASMID MINI PREP
Inoculate 5 ml YT + Amp with single bacterial colony. Incubate at 37 C over night with
shaking.
Label 3 eppendorf tubes for each prep.
Pour 1.5 ml culture into eppendorf tube. Centrifuge for 30 seconds, aspirate and discard
medium, refill tube and repeat. Store remaining culture at 4 C.
Resuspend cell pellet by vortexing in 200 Ál TE + glucose at room temperature.
Add 200 Ál freshly made 0.2 N NaOH/1% SDS. Mix by inverting the tube 5 times, then place
on ice and open tube tops. Incubate 5 minutes (no longer).
Add 200 Ál cold 3 M/5 M potassium acetate (pH approx. 4.8). Mix by inverting the tube 5 times,
then incubate on ice for 5-10 minutes.
Spin in microfuge for 5 minutes.
Transfer to new tube containing 600 Ál phenol: chloroform (2:1). Mix by vortexing, spin 2
minutes in microfuge.
Remove (top) aqueous phase to new tube.
Add 420 Ál isopropanol, mix by vortexing, spin 5 minutes in microfuge.
Pour off supernatant and stand inverted tube on kimwipe to drain.
Add 800 Ál 80% ethanol (-20 C), vortex and spin 5 minutes in microfuge. Pour off supernatant,
drain tube, then dry pellet in speed vac.

  

Source: Auble, David - Department of Biochemistry and Molecular Genetics, University of Virginia

 

Collections: Biology and Medicine