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DNA Extraction for Southern Blot (Meiotic Recombination Intermediates)
- Fix 10 ml cells in 70% EtOH at –20° C (can do 14 ml
at OD ~1.2).
- Wash once with spheroblast buffer (1 M sorbitol, 10 mM NaPO4
pH 7.0, 50 mM EDTA).
- Resuspend in 0.5 ml spheroblast buffer + 3 µl ß-mercaptoethanol
+ 0.5 µl zymolyase (100T 10 mg/ml).
- 15 min at 37°C (or longer until spheroblasted — check
with half-half 2% SDS on slide).
- Spin 3 min at 4000 rpm.
- Resuspend in 0.5 ml lyse solution (50 mM EDTA, 0.3% SDS) + 5
µl protK (20 mg/ml).
- 30 min at 65° C.
- Put on ice.
- Add 0.2 ml 5 M KAc and invert several times.
- 20 min on ice.
- Centrifuge to remove cell debris.
- 3 x phenol chloroform extraction (can rock ~30 min rather than
vortex).
- 1 x chloroform extraction (original protocol used ether —
I use rocker here too).
- 2 x EtOH precipitation (I add 1/10 x 3 M NaOHAc (pH 5.2), mix
then 2 x EtOH, 30 min at –20° C).
- Resuspend (can use ~40 µl 10 mM TrisHCl) and store at
4° C.
- Run 25% total on gel after digest (a lot, but looking for really
rare events).
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