problem in CsCl-EtBrcentrifugement - (Nov/05/2001 )

Hi,Can anyone tell me the condition to use Vetical Ti rotor to replace Ti rotor or NTi rotor in CsCl-EtBr centrifugement to purify a plasmid vector?Thanks a lot!

Wei Liu

I have done 30-60 large scale plasmid preps by the CsCl utracent. method.  I start w/ a 500 ml culture grown in LB for 16-20 hours.  Next, I use the alkali lysis method (10 mls soln. 1 -on ice ~5', 20 mls soln. 2-on ice 5 ', then add 15 mls soln. 3-vortex several times and place on ice 10-15'.  Spin @ 8,000xg 10' and decant supernatent into a 250 ml glass cent. tube-add 90 mls 95% EtOH, incubate @ -20 C for 1 hour.  Then spin @ 8,000xg for 10'.  Decant sups', invert the tube and dry for about 45'.  Add 4.5 mls TE Bfr, 4.5 g CsCl and 360 ul EtBr (from a 10 mg/ml stock).  Mix and fill a sm. ultracentrifuge tube, melt top and ensure that the tube is sealed, balance tube w/ another tube to +/- 0.5 g.  Add to a Vti 65 rotor (or other rotor), tighten cap down and spin @ 47,000xg for 10-16 hours and stop cent-let cent stop slowly.   Take a 16 g needle and vent the top of the tube, take a 18 g needle and extract plasmid band (lower band) w/ visualizing w/ a long wave U.V. light.  Add 1-2 ml's TE, extract until clear w/ Isoamyl alc., ppt. DNA w/ the addition of 1/10 vol. soln. 3 and 2 vol's 95% EtOH.  Inc. @ -20 C for 30' (don't inc. @ -80 C, or the CsCl will ppt. out)-spin @ 10,000xg for 10', wash 1x w/ 70& EtOH.  Dry and resuspend then spect. the DNA.