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Protocols PlasmidExtraction
From 2008.igem.org
Overnight Cultures
Flask with 25mL - 50mL media with corresponding antibiotic
Pick colony with pipette tip/loop and swirl in media
37*C Shake overnight
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Pellet Cells
Transfer overnight cultures to 15mL centrifuge tube (blue cap)
Centrifuge 3000rpm for 6 min
Miniprep
1 Resuspend pellet in 250ul Buffer P1. Transfer to 1.5 mL microcentrifuge tube (by pipeting)
Be sure to vortex (with blue cap tube) thoroughly and that RNAse A was added to Buffer P1
2 Add 250ul Buffer P2 and mix thoroughly by inverting the tube gently 4-6 times
Do not vortex. Maximum reaction time: 5 minutes
3 Add 350ul Buffer N3 and mix immediately and thoroughly by inverting tube 4-6 times
4 Centrifuge for 10 minutes at 13,000 rpm (or 14,000) in a table-top microcentrifuge
5a Prepare the vacuum manifold and spin columns (blue)
5b Apply the supernatant from step 4 to the spin columns by decanting or pipetting
6 Vacuum
7 Wash the spin columns by adding 0.5mL Buffer PB. Vacuum
8a Wash the spin columns by adding 0.75mL Buffer PE. Vacuum
8b Repeat step 8a
9 Transfer the spin columns to a microcentrifuge tube. Centrifuge for 3 minutes
10 Place the column in a clean 1.5 mL microcentrifuge tube
- To elute DNA, add 50ul 2mM Tris-Cl or d.s. H2O to the center of the spin column, let stand for 1 minute, and centrifuge for 1 minute
