Protocol for extracting DNA from ES Cells, starting from the 96-well plate but processing in an eppendorf tube to recover more of the DNA. NOTE- THIS TAKES A LOT OF TIME if you do the whole plate this way!

Lysis buffer: ( 100 ml Recipe )
10 mM Tris-HCl pH7.5 - 0.5 ml of 2M
10 mM EDTA - 2 ml of 0.5M
10 mM NaCl - 0.2 ml of 5M
0.5% (w/v) Sarkosyl - 0.5gm (N-Lauroylsarcosine, Sigma # L-9150)
1mg/ml Proteinase K - add fresh each time (stored in freezer).

1. Add 250µl Lysis Buffer (with proteinase K added) to each well.

2. Incubate the plate in a sealed humid container at 60°ree;C for 1 hour. (Meanwhile, label eppy tubes!)

3. Transfer the contents of each well into a separate 1.5ml eppendorf tube (µ-cent. tube).

4. Continue lysing the cells for 2 more hours. (60°ree;C). (Float tubes in waterbath- don't need tupperware).

5. Add an equal volume (250 µl) of phenol:chloroform (phenol: chloroform: isoamylalcohol, 25:24:1).
(The pH of the phenol:chloroform needs to be ~7.8 - 8.0, or else the DNA will partition into the organic phase).

6. Mix the contents of the tube until an emulsion forms. (Shake by hand for ~1 min, (or vortex ~2sec),because vortexing shears long (genomic) DNA).

7. Centrifuge 5-10 min in a microfuge at TRm. (The phases should be well-separated).

8. Transfer (pipet) the aqueous phase (the top phase) to a fresh tube. (Toss the interface/organic phase-- chemical waste in hood).

9. Repeat steps 5-8 until no protein is visible at the interface of the aqueous and organic phases.

10. Add an equal volume (250 µl) of chloroform and repeat steps 6-8.

11. Optional: Repeat chloroform extraction. (The chloroform extraction removes the traces of phenol).

12. Add 1/25th volume of 5M NaCl (10µl of 5M NaCl for 250 µl ), for a final concentration of 0.2M NaCl. Mix well. (use NaCl rather than other salts because of detergent in lysis buffer).

13. Add exactly 2 volumes of ice-cold ethanol and again mix the solution well.

14. Store the ethanolic solution on ice for 30 min to precipitate the DNA. (can be stored indefinitely at 0°ree;C or at -20°ree;C).

15. Centrifuge at 12,000xg for 10 min, 0°ree;C. (Try 4°ree;C, microfuge at max (16,000), 10 min). (Remember to point tube hinges out, to locate pellet even if it's invisible).

16. Suck off the supernatant (dispo pipet tip, with vacuum flask). Do not disturb the pellet. Vacuum droplets from walls of tube as well.

17. Half fill the tube with 70% EtOH and re-spin for 2 minutes at 4°ree;C.

18. Suck off supernatant as in step 16.

19. Store the open tube on the bench at TRm until the last traces of fluid have evaporated. (i.e.: Air dry).

20. Dissolve the DNA in 30µl (or 50 µl, and use half of sample) of 10mM Tris pH 8.5 (or TE). (pipet around the walls of the tube to dissolve the DNA off the walls of the tube). Let dissolve for at least an hour at 37°ree;C.

21. Restriction digest:
- DNA in Tris pH8.5, from above protocol
- restriction enzyme
- restriction enzyme buffer
- X (BSA if required by enzyme)
- spermidine (to a final conc. of 1mM; stored in freezer)
- (also add: ) DTT to a final conc. of 1mM
Final volume = 40µl or less.
Digest overnight at 37°ree;C.

Next: Run gels; Southern blot and hybridize.
Shirley Reynolds