Boiling Prep for Plasmid DNA - UNC DEPARTMENT OF BIOLOGY
Boiling Prep for Plasmid DNA
1. Spin down cells.
2. Resuspend cells in 350 µL STET buffer.
3. Add 25 µL of 10 mg/mL lysozyme, vortex.
4. Boil 30-40s in 100ºC water bath or 95ºC heat block.
5. Add 10 µL of 10 mg/mL RNase.
6. Centrifuge max speed 15 min.
7. Add supernatant to 400 µL isopropanol. Invert to mix.
8. Pellet 10 min in centrifuge at max speed.
9. Wash pellet with 70% ethanol.
10. Resuspend in water or TE.
STET Buffer
40 g sucrose
25 mL Triton X-100
25 mL 1M Tris, pH 8
50 mL EDTA, pH 8
QS to 500 mL
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