Isolating DNA Fragments

 

<center> <font color="#3333cc"><strong><b><u>Isolating DNA Fragments</u> </b> </strong> </font></center>

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 µl NaI).
5) Melt gel slice in 55¡ water bath.
6) Add 5 µl 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 µl TE pH 7.5. Heat at 55¡ 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 Na2 SO3 (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 Na2 SO3 . Store in dark at 4¡C.

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