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HIDEYO OHUCHI AND SUMIHARE NOTI
- 3M NaCl
- 0.3M sodium citrate
Note: Adjust pH with citric acid
- Solution I (store at RT)
- 50% formamide
- 5x SSC (pH4.5)
- 1% SDS
- Solution III (store at RT)
- 50% formamide
- 2x ssc (pH4.5)
- 1% SDS
- TBST (store at RT)
- 100mM Tris HCI (pH 7.5)
- 150mM NaCI
- 0.1 or 1% Tween-20
- 1.5% Blocking reagent (Boehringer)
Note: Dissolve at 65°C in TBST. Store in aliquots at -20°C.
Embryo powder (optional): Preparation of embryo powder
- 1. Wash appropriate stage embryos in ice-cold PBS.
- 2. Homogenize the embryos in fresh ice-cold PBS.
- 3. Add 4 vol. of ice-cold acetone, mix, and incubate at RT
for 30 mins.
- 4. Centrifuge at 10000 g for 10 mins, remove the supernatant, and then wash the pellet with ice-cold acetone and
spin again.
- 5. Spread the pellet out on a sheet of filter paper.
- 6. Dry the pellet in a disiccator overnight.
- 7. Grind the dried pellet into a fine powder and store in an
air-tight tube at 4°C.
- Anti-DIG-AP (alkaline phosphatase) conjugate, Fab fragment
(Boehringer; store at 4°C)
Note: Do not freeze.
- NTMT (freshly prepared)
- 100mM Tris HCI (pH 9.5)
- 100mM NaCI
- 50mM MgCh
- 1% Tween-20
- AP buffer (freshly prepared)