1. Prepare Lysis Buffer

Lysis Buffer:
12.5 ml SDS 20%
10 ml NaCl 5 M
10 ml EDTA 0.5 M
10 ml Tris 1 M @ pH 7.6
457.5 ml H2O___________
Total: 500 ml

Ratio: 20 μl of 25 mg/ml protease K (-20 °) in 1 ml lysis buffer
For extracting DNA from tail, add 150 microliters lysis buffer (+ protease K) to each tube (using filtered tip).

Example: 10 tubes --- need 1500 μl or 1.5 ml lysis buffer; make extra so ~ 2000 μl or 2 ml .: add 40 μl of protease K or 30 μl protease K for only 1.5 ml

2. Add 150 μl aliquot of lysis buffer (+ protease K) to each tube (using filtered tip).

3. Incubate at 55 ° overnight.

4. Wearing gloves, mix tubes by tapping lightly (in room with cell hood).

5. Centrifuge @ 13,000 rpm for 5 mins @ room Temp.

6. Transfer supernatant to clean, labeled tubes (label tubes on clean paper towels or close tubes to label). Use regular tips and RNA/PCR pipetteman to transfer supernatant. If any of the pellet is taken up by the pipette, re-centrifuge the tube before performing the transfer of supernatant.

7. Add 500 μl isopropanol (using filtered tip) to each tube containing a pellet (do not allow the pipette tip to touch the tubes, shoot in away from the tube (use only one tip for all transfers)). Volume added does not have to be exact.

8. Close tubes, then take to bench to vortex each for 10 sec.

9. Centrifuge at room temperature @ max speed of 13,000 rpm for 5 mins.

10. Remove supernatant and poor into waste cup.

11. Wash with 70% ethanol (500 μl to each tube, then poor out into waste cup).

12. Put open tubes on clean paper towels facing down. Shake out excess liquid and allow to dry for 5-10 mins.

13. Add 150-200 μl TE to each tube using a filtered tip.

14. Set at 37 or 55 ° for 10 to 30 mins

By: Olivia Granillo