Duke University/27 June 2009

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June 27, 2009
Harvest bacterial culture by centrifuging at 8000 rpm (6800xg) in microcentrifuge for 2 min at RT. Decant supernatant and remove remaining medium.

Purification/Mini prep Note: An additional elution step with Elution buffer or water will recover residual DNA from the membrane and increase overall yield by 10-20%.
 * 1) Resuspend pelleted cells in 250 ul Resuspension Solution. Vortex or pipet up and down until no cell clumps remain.
 * 2) Add 250 Lysis Solution and mix thoroughly by inverting tube 4-6 times until solution becomes viscous and slightly clear. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
 * 3) Add 350 ul Neutralization Solution and mix thoroughly by inverting tube 4-6 times.
 * 4) Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
 * 5) Transfer supernatant to spin column by decanting or pipetting. Avoid disturbing or transferring white precipitate.
 * 6) Centrifuge for 1 min. Discard flow-through and replace column in collection tube.
 * 7) Add 500 ul Wash Solution to spin column. Centrifuge for 30-60s and discard flow-through.
 * 8) Repeat step 7.
 * 9) Centrifuge for 1 min to remove residual Wash Solution.
 * 10) Transfer spin column into fresh 1.7 ml microcentrifuge tube. Add 50 ul Elution Buffer to center of spin column membrane. Do not touch pipette tip to membrane. Incubate 2 min at RT and centrifuge for 2 min.
 * 1) Discard column and store purified plasmid DNA at -20°C.