Electroelution in "V" Apparatus
(Original protocol by Andy Hoyt at JHU)
(modification by Maier and Fambrough Labs)
1. Make 500 ml elution buffer * : 10 mM Tris-HCl
pH 8.0, 1 mM EDTA, 5 mM NaCl.
De-gas (not the artist) for ~ 10 min. in a sidearm flask on the aspirator.
2. On all 4 sides, tape the electroelution apparatus to the bench.
Fill the apparatus with buffer to cover the well holes but not enough to flow over bridge.
3. Make sure that the gate is open.
Cut out gel slice containing the band of interest. Cut as small a piece as possible.
4. Place gel slice in gel slice well on bridge.
5. Flush out "V" with Pasteur pipette.
6. Carefully dispense 100 µl of 7.5 M Ammonium Acetate into "V".
7. Slide gel slice very close to "V"
slot. Run at 150 volts for 20 minutes.
(Note: running too long can decrease % recovery!)
8. When complete, turn off power supply, make sure to close bridge gate. Remove gel slice and check under UV light for unrecovered DNA.
9. Lower buffer from each side of reservoir (below "V" mouth). Remove buffer from side opposite the gate first.
10. With a Pasteur pipet, remove excess buffer from gel slice well and the hole in gate, while the gate is still closed.
11. Open gate and remove acetate cushion and DNA (~200 µl) with special pipette tip (which is reusable). Put the DNA in a 1.5 ml tube.
12. Rinse the "V" with 150 µl elution buffer and add this to the DNA in the 1.5 ml tube.
13. Add 50 µl 7.5 M ammonium acetate to pooled DNA sample (bringing it to ~400 µl).
14. Add 1 µl of carrier glycogen ** (stock of 20 mg/ml) to the DNA.
15. Add 800 µl EtOH and preciptate as usual (-70° C for 30 min. or -20° C overnight).
16. Spin the DNA for 10 minutes, wash pellet with 70% EtOH, and resuspend DNA in a small volume of TE or water.
** Glygcogen: Boehringer Mannheim #901 393
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