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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