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Star Republic: Guide for Biologists

Extraction of low MW DNA fragments

1. Wash cells (105-106) with PBS, harvest, and transfer to 1.5 ml microcentrifuge tube.

2. Resuspend cell pellet in 0.5 ml of the lysis buffer (5 mM Tris-HCl pH 8.0, 20 mM EDTA, 0.5% Triton X-100).

3. Incubate on ice for 20 minutes.

4. Centrifuge at maximum speed for 30 minutes.

5. Transfer the supernatant to a fresh tube.

6. Extract with 0.5 ml of phenol:chlorofrom:isoamyl alchohol (25:24:1).

7. Add 1/10 volume of 3M sodium acetate and precipitate with 2 volumes of ethanol.

8. Centrifuge to pellet DNA.

9. Wash the pellet with 70% ethanol and air dry.

10. Resuspend the DNA in 30-50 ml of TE/10 mg/ml RNase A.

11. Detect the DNA ladder by running on a 1.2% agarose gel.