Genomic DNA Miniprep
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Genomic DNA Miniprep
- grow 5 ml cells over night at 30 °C
- spin, wash once with 1 ml H2 O
- resuspend in 500 µl lysis buffer
- add acid washed glass beads to 1.25 ml
- vortex 2 min
-
recover liquid phase with blue tip or
punch whole into the bottom of the tube and centrifuge into another tube - add 275 µl 7 M Ammonium acetate pH 7.0
- incubate 5 min at 65 °C , then 5 min on ice
- add 500 µl Chloroform , vortex, spin 2 min in microfuge
- take supernatant and precipitate with 1 ml Isopropanol
- incubate 5 min at RT , spin 5 min
-
wash pellet with 70 % EtOH , dry and dissolve in 50 µl H2 O
- for Southern : digest 5 µl DNA
- for PCR : use 0.5 to 1 µl DNA
Buffers:
lysis buffer:
100 mM Tris pH 8.0; 50 mM EDTA; 1 % SDS.
[for 50 ml : 5 ml 1 M Tris; 5 ml 0.5 M EDTA; 5 ml 10 % SDS]
