Paul.xu at 14:01, 22 March 2016

2016-03-22T14:01:03Z

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#Grow bacterial (''E. coli'') culture in LB medium with appropriate antibiotics at 37 °C overnight (O/N) with shaking. For >10 copies plasmid, 3 ml cell culture is usually enough.
#Spin down cell culture.
#Discard the supernatant.
#Add 200 μl of resuspension solution ('''P1 buffer''') into each tube, and vortex to completely resuspend cell pellet
#Add 200 μl of lysis solution ('''P2 buffer''') and mix by gently inverting the tube 5-6 times. The solution should quickly turn transparent and become more viscous indicating bacterial lysis has taken place.
#Add 200 μl of neutralizing solution ('''P3 buffer''') and mix by inverting the tubes several times. At this point bacterial chromosomal DNA is usually seen as a white precipitate.
#Transfer to a microcentrifuge tube
#Centrifuge at high speed for 2X2.5 min.
#Carefully transfer the supernatant (try to not disturb the white precipitate) to a new labeled 1.5-ml eppendorf tube
#Add 2.5-3 volume of 200-proof cold ethanol (stores at -20 °C) to each tube and mix by inverting the tubes a few times.
#Spin down plasmid DNA precipitate (transparency pellet) at high speed for 10 min.
#Discard the supernatant and remove the remaining liquid as much as possible by leaving the tube upside-down on a piece of paper towel, then keep the tubes in a tube holder and air dry for 10-20 min. To dry faster, keep tubes at 37 °C heat blocker. DNA precipitate turns white when dry.
#Resuspend the DNA pellet with 50 μl TE. Completely dissolve the pellet by pipetting solution several times.
[[Category:Protocols]]

Miniprep-Alkaline Lysis - Revision history

Paul.xu at 14:01, 22 March 2016