High melt crush procedure for extraction of DNA from an agarose gel

1. Pour a 0.8% agarose gel
- 100ml of 0.5X TAE and 0.8g of agarose
- Let gel cool for about 1 hr.
2. Run samples overnight
- 130 volts for 10 min and then reduce voltage to lowest setting (~20 volts)
3. Stain gel for 10 min
- 200ml of fresh 0.5X TAE and 10ul of ETBR
- Prepare staining solution the night before
4. Excise bands and place in eppindorf tubes
5. Weigh excised bands (1g =1ml)
6. If necessary add 1X TE to bring total volume to 500ul or 0.5g (agarose + TE)
7. Crush agarose with pestle
- Rinse pestle between tubes with ETOH and then de-ionized water
8. Add 500ul of saturated phenol (pH~7.9)
9. Vortex well
10. Place at -80˚C until frozen (usually 5min)
- This is a good stopping point if necessary
11. Thaw tubes at 37˚C for ~5min
12. Vortex well and place at -80˚C until frozen (usually 5min)
13. Thaw tubes at 37˚C for ~5min
14. Mix well and spin at top speed (13,000rpm) for 10min
15. Remove the aqueous phase (top)
16. Add 500ul of chloroform
17. Vortex well
18. Spin at top speed (13,000rpm) for 2min
19. Remove the aqueous phase (top); discard chloroform
20. Add 2X volume salty ETOH
- Dilute a concentrated solution (6M Ammonium Acetate, 0.2M Magnesium Chloride) 1/20 (5ml in 95ml ETOH)
21. place at -20˚C for 10min
22. Spin at top speed (13,000 rpm) for 10min (4˚C)
23. Discard supernatant (be careful not to lose pellet!!)
24. Air dry for 5min
25. Resuspend pellet in 10ul of 10mM Tris
26. Run out 1ul on an agarose gel to quantitiate