- grind the fly in 30 µl of buffer A
- spin for one minute
- remove the supernatant (contains RNA), suspend the pellet
(with nuclei) in 200 µl buffer A
- spin for one minute, discard supernatant
- suspend the pellet in 18 µl of buffer B containing
proteinase K
- add 2 µl 10% SDS, mix quickly
- incubate one hour at 37°C, (or longer, upto 4 h)
- add 3 µl 3 M NaCl
- add 10 µl phenol/chloroform, mix
- spin, transfer 20 µl to new tube
- add 50 µl ethanol, mix
- spin 5 min, wash pellet with 200 µl 70% ethanol
- briefly dry in speed-vac ( one minute)
- dissolve the pellet in 20 µl TE
- use 1 µl for PCR
After use treat the pestles with bleach. Rinse with water and store
them.
buffer A:
30 mM Tris/HCl [pH 8], 100 mM NaCl, 19 mM EDTA, 0.5% Triton X-100
3.3 ml 3 M NaCl
3 ml 1M Tris/HCl [pH 8]
3.8 ml 0.5 M EDTA
2.5 ml 20% Triton
ad 100 ml water
buffer B:
30 mM Tris/HCl [pH 8], 100 mM NaCl, 19 mM EDTA
3 ml 1 M Tris/HCl [pH 8]
3.3 ml 3 M NaCl
3.8 ml 0.5 M EDTA
ad 100 ml water
buffer B with proteinase K
prior to use add 1 µl proteinase K (20 mg/ml) to 100 µl buffer B |