Alkaline Lysis Minipreps
Inoculate 5 ml overnights of LB + 50 ug/ml Ampicillin
- 1 ml of Cells to eppendorf tube
- spin 40 seconds, full speed
- discard supernatant (aspirate)
- resuspend cells in 100 ul GTE
- incubate 5 min. RT
- add 200 ul NaOH/SDS
- mix by inversion
- incubate 5 min on ice
- add 150 ul 5 M KOAc pH 4.8
- mix by inversion
- incubate 5 min on ice
- spin 2 min
- remove 410 ul to new tube
- add 1 ml 100% ethanol
- incubate on ice 5 min
- spin 5 min
- remove sup
- add 500 ul 70% ethanol (-20 C)
- spin 5 min
- remove sup
- spin vac 5 min.
- resuspend in 20 ul TE
Use 1 to 5 ul for digests.
RNaseA may be added to GTE to 125 ug/ml, or mix 20 to 100 ng RNaseA per reaction in
with the loading dye.
For sequencing, add 80 ul TE and phenol/chloroform extract, then ethanol precipitate.
Resuspend in 20 ul.
GTE
50 mM Glucose (40% is 2.2M)
25 mM Tris-HCl pH 8
10 mM EDTA
NaOH/SDS
1% SDS
0.2 N NaOH
GTE Calculator - Simple JavaScript. Enter the Total Volume of GTE you would like
to make and click anywhere, and the volume of each component will be filled in.
Chris Seidel October 1998