Jeff's CMBL miniprep

For many labs

Before beginning, add 95% ethanol to Plasmid Wash Buffer 4:1 Also, once RNAse has been added to P3 (yellow), keep it at 4C.

Protocol

• Spin 0.5 - 5 mL bacterial culture in 1.5 mL tube 20 sec full speed

• Add 200 uL P1 (red), resuspend pellet

• Add 200 uL P2 (green); mix by inverting 2-4 times. Clear, viscous, purple solution indicates cell lysis

• Add 350 uL P3 (yellow). Mix thoroughly. DO NOT VORTEX. Turns yellow when neutralization is complete.

• Incubate RT 1-2 min

• Spin 2 min

• Put column into collection tube, transfer supernatant. DO NOT DISTURB GREEN PELLET (I found it wasn't green, but OK)

• Spin 30 sec

• Discard flow through, put column back into emptied collection tube

• Add 200 uL Endo-Wash Buffer to the column, spin 30 sec

• Add 400 uL Plasmid wash buffer to the column, centrifuge 1 min

• Transfer column to clean 1.5 mL microcentrifuge tube

• Add 30 uL DNA Elution Buffer

• Spin 30 seconds. Plasmid DNA is in the collection tube

Students need per reaction¹

• 1 mL (?) overnight culture of transformed cells (or spun down pellet of ON culture)

• 1 column, 1 2-mL collection tube

• sterile 1.5 mL tube

• ~200 uL P1 (red)

• ~200 uL P2 (green)

• ~350 uL P3 (yellow)

• ~200 uL Endo-Wash-Buffer

• ~400 Plasmid Wash Buffer

• ~30 uL DNA Elution Buffer