Gel Extraction (from 25 μl PCR Reaction)

Bring Tris-buffered phenol and Chloroform/Phenol/Isoamyl Alc to room temp

1)      Cut out bands from gel under UV light.

2)      Melt gel at 70°C.

3)      Add an equal volume of dH₂O to 250 μl total volume.

4)      Add 250 μl of Tris buffered phenol (RT), vortex, spin max speed, 5 minutes.

5)      Remove upper aqueous layer, transfer to new tube.

6)      Repeat steps 4-5 one time.  Upper layer transferred should be clear!

7)      Add an equal amount of phenol:chloroform.  Vortex, spin max speed 5 minutes.

8)      Transfer upper aqueous layer to new tube and precipitate upper layer with 2.5μl/100μl sample 5M NaCl.

9)      Add 1 μL glycogen.

10)  Add 2.5x volume ICED EtOH.

11)  Precipitate at -20°C overnight or -80°C for 1-2 hours.

12)  Spin 15 minutes at 4°C to pellet.

13)  Discard supernatant, rinse pellet once with 70% EtOH. (100 μl 70% EtOH)

14)  Dry excess EtOH from tube, resuspend pellet in ~ 10 μl H₂O (DNA H₂O).

15)  Run 1 μl on gel to check for DNA recovery:

        1 μl DNA + 2 μl 6X load dye + 9 μl dH₂O