Alkaline Lysis Mini-Prep
Dominik Haudenschild, Monday, October 06, 1997
1: Pellet 1.5ml of overnight bacterial culture in Eppendorf tube, discard sup.
2: Resuspend completely in 100µl of cold Resuspension Buffer by vortexing
3: Add 200µl Lysis Buffer, mix by inverting tube gently
4: Add 150µl Precipitation Buffer, mix by short vortex,
5: Incubate on ice for 3-5 minutes
6:Centrifuge top speed for 5 minutes
7: Put Supernatant into fresh 1.5ml tube, discard pellet (has bacterial crap)
8: Precipitate plasmid DNA by with 950µl ethanol
9: Pellet DNA by 5-10 minutes top speed in centrifuge
10: Remove supernatant, wash pellet with 75% ethanol, then air-dry pellet
11: Redissolve DNA in 50µl TE with 20µg/ml DNAse-free Pancreatic RNAse
12: Store DNA in -20
|
Resuspension Buffer |
Lysis Buffer |
Precipitation Buffer |
|
50mM Glucose |
0.2N NaOH |
60ml 5M Potassium Acetate |
|
25mM Tris pH 8.0 |
1% SDS |
11.5ml Glacial Acetic Acid |
|
10mM EDTA |
28.5ml Water |
DNA is suitable for restriction enzyme digestion
DNA is NOT suitable for automated sequencing or ligations