Biology Reference
In-Depth Information
8. Blocking buffer
Milk Blocker (Bio-Rad # 170-6404)
2.5 g
TN
to 50 mL
Make in sterile plastic 50 mL Falcon tubes. Suspend by vortexing.
9. 0.93M Tris, pH 9.5
1M Tris, pH 7.5 (Fisher, BP1757500)
500 mL
10M NaOH
43.5 mL
Dispense aliquots into sterile 50-mL Falcon tubes and freeze at
20
C. May be
thawed in 37
C incubator prior to use. (We have found pH adjustment of the
RNase-free Fisher stock to be a more convenient way to make this buffer than to
make 1M Tris pH 9.5 by other means.)
10. Tris, NaCl, MgCl
2
, (pH 9.5) (2
TNM),
0.93M Tris (pH 9.5) (see above) 100 mL
2M NaCl 50 mL
1M MgCl
2
50 mL
DEPC-ddH
2
O to 500 mL
Autoclave for 15 min at 121
C, 15 lb/in
2
. Dispense aliquots into 50 mL Falcon
tubes. Store at
20
C. Thaw the morning that it is needed. If a white precipitate
occurs, heat in a 65
C waterbath and vortex, then cool before use.
To 25 mL of 2
TNM, add 25 mL DEPC-ddH
2
O. This is used as the last wash
prior to development. The other 25 mL aliquots are used to make the Developer
solution.
11. Nitro Blue Tetrazolium (NBT)
NBT is resuspended to 100 mg/mL in 70% dimethyl formamide and stored in
aliquots at
20
C.
12. 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP)
BCIP is resuspended to 50 mg/mL in anhydrous dimethyl formamide and stored
in aliquots at
20
C.
13. 2
Polyvinyl alcohol (PVA)
PVA (Fluka 40-88, Sigma #81386)
50 g
DEPC-ddH
2
O
to 500 mL
This solution takes very long to go into solution. It is best to start with freshly
autoclaved (warm) DEPC-ddH
2
O and add PVA to a sterile container. Incubate at
65
C for several days, stirring often with a 25 mL plastic serological pipette; A
stir bar is not recommended. When it is fully in suspension, the solution will
have the consistency of glycerol. Store at room temperature.
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