Extracting DNA as a fiber/strand - Dumb question! Need practical help. (Oct/30/2006 )
QUOTE (ML1975 @ Nov 1 2006, 10:45 PM)
OK..here's a quick recipe. Make XS buffer:
0.5 g Potassium ethyl Xanthogenate (Fluka 140-89-6)
10 mL 4M Ammonium acetate
5 mL 1M Tris-HCl pH 7.4
2 mL 0.45M EDTA
2.5 mL 20 % SDS
Water to 50 mL
Pellet 1-2 ml of bacterial culture in a 2 mL eppendorf
Remove supernatant and resuspend pellet in 1 ml XS buffer
Incubate @ 70 degrees for 1 hour
Vortex tube for 10 sec and incubate on ice for 30 min
Centrifuge at 14, 000 rpm for 10 minutes
Carefully remove supernatant to a new tube and add 1 volume of isopropanol
Invert tube a few times, DNA will precipitate as a thread
spool out with a tip or tooth pick
Wash spooled DNA in a tube containing 70 % ethanol
Resuspend DNA in 200 ul of dH20
0.5 g Potassium ethyl Xanthogenate (Fluka 140-89-6)
10 mL 4M Ammonium acetate
5 mL 1M Tris-HCl pH 7.4
2 mL 0.45M EDTA
2.5 mL 20 % SDS
Water to 50 mL
Pellet 1-2 ml of bacterial culture in a 2 mL eppendorf
Remove supernatant and resuspend pellet in 1 ml XS buffer
Incubate @ 70 degrees for 1 hour
Vortex tube for 10 sec and incubate on ice for 30 min
Centrifuge at 14, 000 rpm for 10 minutes
Carefully remove supernatant to a new tube and add 1 volume of isopropanol
Invert tube a few times, DNA will precipitate as a thread
spool out with a tip or tooth pick
Wash spooled DNA in a tube containing 70 % ethanol
Resuspend DNA in 200 ul of dH20
Thanks a lot! So the basic suggestion I'm getting from everyone is to grow my own bugs, not do something that would shear the DNA, kill the RNA if I need to, and then precipitating DNA gives nice strands - thanks a lot, I'll let you know how it goes!
-Foxbat-