Isolating DNA fragments

Materials:

0.8 % agarose gel in 1x TAE

Digested DNA

Glass Milk

NaI solution

New Wash

Procedure:

1) Run digested DNA out on agarose gel slowly (70 V on BioRad gel)

2) Use long wave UV lamp to visualize bands. Cut out band with scalpel. Cut smallest possible piece.

3) Put gel slice in an eppendorf tube and weigh to figure out volume of gel slice. (empty tube approx. 1 g).

4) Add 3 vol of NaI solution (gel slice is usually ~200 mg, therefore, add 600 ul NaI).

5) Melt gel slice in 55 degree water bath.

6) Add 5 ul glass milk and mix (vortex glass milk vigorously to resuspend before adding to DNA mx). Incubate on ice ~5 min.

7) Spin down glass milk quickly. Decant. Add 1 ml New Wash and vortex. Do a total of 3 washes.

8) Spin down hard to pellet glass milk. Aspirate off liquid and air dry.

9) Resuspend pellet in 30-50 ul TE pH 7.5. Heat at 55degrees for 5 min.

10) Spin down glass milk and save supernatant. This is your DNA.

NaI solution

Mix 90.8 g NaI and 1.5 g Na2SO3 (note: sulfite!) to a final volume of 100 ml (cover with foil while stirring, light sensitive). Filter through #1 Whatman paper add 0.5 g Na2SO3. Store in dark at 4degreesC.

New Wash for 500 ml

50% EtOH 250 ml

0.1 M NaCl 2.92 g

10 mM Tris 7.5 5 ml 1M

1 mM EDTA 1 ml 0.5 M