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:
-
Aspirate media from 100 mm dish of confluent ES cells.
-
Rinse and aspirate dish twice with 10 ml PBS.
-
Add 3 ml Trypsin/EDTA and incubate @ 37°C for 5 min.
-
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.
-
Raise volume to 10 ml and centrifuge @ 1000 rpm for 5 min @
RT.
-
Aspirate media and re-suspend in 10 ml PBS to wash cells.
-
Centrifuge @ 1000 rpm for 5 min @ RT.
-
Aspirate PBS and freeze pellet @ -70°C until ready to
lyse.
-
Re-suspend cell pellet in 3 ml lysis buffer (w/1 mg/ml proteinase K
added just prior to use).
-
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:
-
To the 3ml of lysis buffer, add 5 ml DNA Phenol w/glass
pipette.
-
Mix on "Nutator" for up to one hour. Centrifuge @ 1000 rpm
for 15min @ RT.
-
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.
-
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.
-
Repeat phenol extraction until no white precipitate (protein) is
visible (usually 2- 3 times).
-
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.
-
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.
-
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:
-
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
-
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.
-
Take the Pasteur pipette, twirl in the tube and wrap the DNA around
it.*
-
Carefully move pipette to labeled tube containing 6-9 ml 70%
ethanol and with a 2nd pipette remove the DNA from the first
pipette.
-
Centrifuge @ 1000 rpm for 5 min @ RT.
-
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.
-
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.
-
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