Protocols PlasmidExtraction

Overnight Cultures Flask with 25mL - 50mL media with corresponding antibiotic Pick colony with pipette tip/loop and swirl in media 37*C Shake overnight _____________________________________________

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

Protocols