Preparation of genomic DNA from ES cells
6/8/04
1. For a 24 well plate, pour off medium, then add 300uL Tail Lysis Buffer to each
well. Wrap plate in parafilm to reduce evaporation. Leave O/N in warm room,
then at RT until ready to process. Lysates are stable for 18 months at RT.
2. Add 1.5uL RNAseA to each tube, changing tips in between samples. (RNAseA is
stored in the undercounter frige). Invert the tubes 25X to mix solution. Incubate in
beads in the warm room at 37C for 15’ (or up to 60’).
3. Cool sample to RT for 1’ on ice. Add 100uL protein precipitation solution to each
tube. Vortex at high speed for >20 seconds to thoroughly mix. Chill on ice for 5’.
Spin max speed for 3’ to precipitate proteins. If any residual protein in supe,
vortex, chill, and spin again. Do not pour protein into the next tube.
4. Decant supe into a fresh Eppendorf tube containing 300uL 100% isopropanol.
Carefully label the tubes with the number for each tail. Invert the tubes 50X to
mix solution.
5. Spin top speed for 3’ to precipitate DNA. A small white pellet should be visible.
Pour off isopropanol and dry tubes upside down on paper towel for 5’.
6. Add 300uL 70% EtOH to each tube and invert tubes a few times to wash DNA.
Spin 3’ at top speed to anchor pellet.
7. Pellet may be very loose, so do not let tubes sit after spin and carefully pipette off
EtOH. Air dry on paper towel for 15’. If all EtOH is not gone (and if you can see
the pellet), aspirate or pipette out the excess carefully avoiding the pellet.
8. Once all EtOH is removed, add 20uL DNA hydration solution. Hydrate DNA for
1 hour at 65C. Tap tube periodically to help resuspend DNA. Spin down
evaporated solution and gently pipette DNA to fully resuspend. Leave DNA O/N
at RT to resuspend. Store at 4C. On spec you expect 250-500ug/mL (5-10ug DNA
total).
9. This is enough genomic DNA for ONE southern and a PCR reaction or two. If
you want to do PCR, do that first, then cut DNA in a 25uL volume total, 6 hours
to overnight. Add 5uL 6X DNA SB, and run 0.7% agarose gel.
Buffers:
Tail Lysis Buffer:
25mM TrisCl pH 8.0
10mM EDTA pH 8.0
1% SDS
for 500mL
6.25mL 2M Tris pH8.0
10mL 0.5M EDTA pH8.0
25mL 20% SDS
RNAseA
Dissolve to 4mg/mL in ddH20. Boil 15’ to kill DNAses. Store 4C.
Roche cat# 109 126
Protein Precipitation Solution
7.5M Ammonium Acetate
289.05g Ammonium acetate in 150mL ddH2O, make to 500mL
DNA hydration solution (TE)
10mM Tris pH 7.4
1mM EDTA pH 8.0
If in doubt, remember
(final concentration)(final volume) = (initial concentration)(initial volume)