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DNA Purification - DEAE Nitrocellulose Membranes.

Jeff Lawrence (P01)

1. Separate DNA fragments in an agarose gel cast with 0.5 mg/mL Ethidium bromide. Locate bands with a hand-held long-wave UV lamp.

2. Slice the gel with a razor blade above and below the bands of interest. Insert the membrane into the gel at each slice, avoiding air bubbles between the gel and the membrane. The membranes above the bands of interest prevent contamination with higher molecular weight DNA.

3. Electrophorese at high voltage to run the DNA onto the membrane.

4. Remove the membrane with the bands of interest and verify DNA with long-wave UV. Discard membranes placed above the bands of interest.

5. Rinse the membranes in Low Salt Buffer to remove agarose.

6. Place membrane in 1.5 mL tube. Add 150 ml High Salt Buffer.

7. Incubate at 70 C for 20 min. Remove supernatant and place in a fresh tube.

8. Add 150 ml High Salt Buffer to the membrane. Incubate at 70 C for 10 min.

9. Combine the supernatant with that removed in Step (10). Add 300 ml Buffer Saturated Phenol.

10. Mix well by vortexing. Centrifuge for 15 min at 4 C. Remove the aqueous phase being careful NOT to remove ANY of the interface, which contains fibers of DEAE membrane.

11. Add 300 ml 24:1 Chloroform:Isoamyl alcohol. Mix well by shaking.

12. Centrifuge 2 min at 4 C. Remove the aqueous phase. Add 5 ml 1% linear polyacrylamide carrier and mix well.

13. Add 700 ml 95% ethanol. Incubate at -20 C for 2 hr.

14. Centrifuge for 15 min at 4 C. Discard supernatant. Add 500 ml 70% ethanol. Mix well to dislodge the pellet.

15. Centrifuge for 2 min. Discard supernatant. Air dry.

16. Resuspend DNA in 50 mL TE.