DNA lysis, extraction and isolation from ES cells

DNA Lysis

Reagents, supplies and equipment:

• PBS
• Trypsin/EDTA
• cES
• Lysis buffer w/Proteinase K @ 1 mg/ml

Note: This protocol may be scaled down for smaller cell pellets, e.g., one 60 mm dish of cells may be lysed and extracted in half the volumes used for a 100 mm dish.

Procedure:

1. Aspirate media from 100 mm dish of confluent ES cells.
2. Rinse and aspirate dish twice with 10 ml PBS.
3. Add 3 ml Trypsin/EDTA and incubate @ 37°C for 5 min.
4. Add 3 ml cES media to inactivate trypsin.  Pipette vigorously up and down to disaggregate cells, then move entire volume to a 15 ml conical tube.
5. Raise volume to 10 ml and centrifuge @ 1000 rpm for 5 min @ RT.
6. Aspirate media and re-suspend in 10 ml PBS to wash cells.
7. Centrifuge @ 1000 rpm for 5 min @ RT.
8. Aspirate PBS and freeze pellet  @ -70°C until ready to lyse.
9. Re-suspend cell pellet in 3 ml lysis buffer (w/1 mg/ml proteinase K added just prior to use).
10. Invert tube 2-3 times and place in 60°C H₂0 bath or hybridization oven overnight.

Phenol & chloroform extractions

Reagents, supplies and equipment:

• DNA phenol (pH8)
• Chloroform/Isoamyl alcohol @ 24:1
• "Nutator" (small rocking device for mixing)

Procedure:

1. To the 3ml of lysis buffer, add 5 ml DNA Phenol w/glass pipette.
2. Mix on "Nutator" for up to one hour.  Centrifuge @ 1000 rpm for 15min @ RT.
3. Carefully and slowly with a cut pipette tip on a P1000 remove the top layer, making sure not to pick up any of the white protein layer, and place in new clearly labeled conical tube.
4. Add 5 ml DNA phenol w/glass pipette to this clear layer and mix ~15 min Centrifuge @ 1000 rpm for 15 min @ RT, and remove top layer again.
5. Repeat phenol extraction until no white precipitate (protein) is visible (usually 2- 3 times).
6. Add 2.5 ml phenol + 2.5 ml chloroform/isoamyl alcohol (24:1), mix ~ 15 min, centrifuge @ 1000 rpm for 15min @ RT and remove top layer again.
7. To remove the residual phenol, add 5 ml chloroform to DNA layer, mix 15 min, centrifuge @1000 rpm for 30 mi.n@ RT and remove the top layer.  Repeat if necessary.
8. Store supernatant @ 4°C O/N or proceed to isolation steps.

Isolation of DNA

Reagents, supplies and equipment:

• Ethanol: 95% and 70%
• Glass Pasteur pipettes w/sealed end
• 3 M sodium acetate, pH 5.2
• Nuclease-free water, or TE

Procedure:

1. Add 0.1 x volume 3 M sodium acetate (300 µl in this case) to the tube with 3 ml from the last chloroform extraction 
2. Add 2 x volume 95% ethanol (6 ml in this case) and quickly invert the tube 2-3 times.  You should see the stringy white DNA precipitate.
3. Take the Pasteur pipette, twirl in the tube and wrap the DNA around it.* 
4. Carefully move pipette to labeled tube containing 6-9 ml 70% ethanol and with a 2nd pipette remove the DNA from the first pipette.
5. Centrifuge @ 1000 rpm for 5 min @ RT.
6. Carefully pour ethanol into the sink (or a second tube if you are concerned about losing the pellet), making sure the DNA pellet does not dislodge.
7. Blot excess ethanol with paper towel or Kimwipe and allow the tubes to air dry 10-20 min.  Do not allow the DNA pellet to dry completely, otherwise it will be very difficult to dissolve.
8. Re-suspend in 200 µl of nuclease-free water or TE and store @ 4°C.

*Alternatively, centrifuge @ 1000 rpm for 5 min to pellet the DNA, then wash pellet in 70% EtOH, centrifuge again and pour off ethanol.

Source: ES Cell Core Lab

Revised: 12/18/03 by Susan Hipkens