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Genomic DNA Mini-prep
Adapted for Fastprep machine
Solutions
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
Procedure
- Grow 5 ml cells overnight at 30° C.
- Spin, wash once with 1 ml H2O.
- Resuspend in 500 µl lysis buffer.
- Transfer to a screw-tube with acid washed glass beads.
- Fastprep at 6.0 speed for 20 min.
- Recover liquid phase with blue tip 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 room temperature, then spin 5 min.
- Wash pellet with 70% EtOH, dry and dissolve in 50 µl H2O.
For Southern, digest 5 µl DNA.
For PCR, use 0.5-1 µl DNA. |
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