In Situ Hybridization Solutions
5X NTE
1 M Tris pH 7.5 5 ml
0.5 M EDTA pH 8.0 5 ml
5 M NaCl 5 ml
35 ml ddH2O (DEPC-treated)
Divide into aliquots; store at -20°C.
Proteinase K, 10 mg/ml stock
Proteinase K [Sigma #P2308] 100 mg
DEPC ddH2O 9 ml
o In sterile 15 ml tube, mix the PK and sterile ddH2O by inversion
o Bring up the volume to 10 ml with sterile ddH2O
o Divide into 600 ml aliquots, store at -20°C.
Denhardt’s Solution (50X)
Ficoll (Fisher, #BP525) 5 g
Polyvinylpyrrolidone (Fisher, #BP431) 5 g
BSA (Sigma, #B4287) 5 g
Bring up volume to 500 ml with ddH2O
Divide into aliquots; store at -20°C.
Hybridization juice
Reagent Amount [Final ]
UltraPure Formamide [Sigma #47671] 25ml 50%
1M Tris/HCl, pH 7.6 500ml 10mM
tRNA, 10mg/ml [Sigma #R8508] 1ml 200mg/ml
50X Denhardt’s 1ml 1X
50% dextran sulfate [Millipore #S4031] 10ml 10%
5 M NaCl stock 6ml 600mM
10% SDS 1.25ml 0.25%
DEPC ddH2O 5.25ml
_____
Total volume: 50ml
- Divide into aliquots (will need 4-5 mL for 50 slide experiment), store @ -20°C
1 M Dithiothreitol (DTT)
10 mM sodium acetate, pH 5.2
3 M sodium acetate, pH 5.5 100 ml
o pH to 5.2 with HCl
o Bring up volume to 30 ml with DEPC ddH2O
1 M DTT
DTT [Promega #V3151] 3.09 g
10 mM sodium acetate, pH 5.2 bring volume up to 20 ml
o Aliquot into 200 ml aliquots, store at -20°C.
RNase A (Ribonuclease)
10 mM Tris/15 mM NaCl Buffer
1 M Tris, pH 7.6 100 ml
5 M NaCl 30 ml
o Bring up volume to 10 ml with sterile ddH2O
RNase A, 20 mg/ml stock
10 mM Tris/NaCl buffer 10 ml
RNase A [USB, #21195] 200 mg
o In sterile 15 ml tube, mix the RNase A and 10mM Tris/NaCl by inversion
o Bring up the volume to 10 ml
o Divide into 900 ml aliquots, store at -20°C.
Photoemulsion
o Use Kodak Autoradiography Emulsion NTB 2, available from VWR Scientific [#IB8895666].
o Aliquoting Emulsion (in darkroom!):
o When emulsion arrives, divide into 5 equal parts and put into small, opaque bottles.
o Wrap each bottle with 3 layers of aluminum foil and label with content and expiration date.
Updated 11/30/09 by RK