Oneill Lab:WMISH Chemicals

Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology

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WMISH Solutions

Post-Fix

This solution must be made fresh. Glutaraldehyde is a potent fixative, and should only be used in a chemical fume hood. Do not dispose of excess fix or glutaraldehyde solution in sink.

Post-fix Solution

Ingredients	5ml	10ml	15ml	20ml
37% Formaldehyde	915.5 μL	1.831 mL	2.7465 ml	3.662 ml
25% Glutaraldehyde	20 μL	40 μL	60 μL	80 μL
PBS + 0.1% Tween20	4.0645 mL	8.121 mL	12.1935 mL	16.258 mL
1M NaOH	4.5 μL	9 μL	13.5 μL	18 μL

Hybridization Buffer

Hyb buffer should be made fresh for each hybridization. Excess can be stored at -20°C for use in post-hyb washes. Always preheat to 70°C before use.

Hybridization Buffer

Ingredients	[Final]	50mL
Formamide	50%	25mL
DEPC 20x SSC	1.3x	6.5 mL
DEPC 0.5M EDTA	5mM	0.5 mL
10mg/ml tRNA	50 μg/mL	250 μL
Tween 20	0.2%	101 μL
10% CHAPS	0.5%	2.5 mL
50 mg/ml Heparin	100 μg/mL	100 μL
DEPC Water		18.3 mL

TBST

After all materials combined, solution should be filter sterilized.

1x TBST

Ingredients	500 mL
5M NaCl	13.69 mL
1M KCl	1.34 mL
1M Tris pH 7.5	12.5 mL
Tween 20	505 μL
DEPC Water	to 500 mL

MABT

After all materials combined, solution should be filter sterilized.

1x MABT

Ingredients	500 ml
1M Tris pH 7.5	50 ml
5M NaCl	15 ml
Tween20	505 μL
DEPC-treated H2O	to 500 ml

2% Blocking Solution

2% Blocking Solution

Ingredients	50 ml
Blocking reagent	1g
1M Tris pH 7.5	5 ml
5M NaCl	1.5 ml
DEPC-treated H2O	43.5 ml

• Dissolve components at 63°C with shaking.
• Store at 4°C

Genius 3

After all materials combined, solution should be filter sterilized.

Genius III Solution

Ingredients	150 ml
1M Tris pH 9.5	15 ml
1M MgCl2	7.5 ml
5M NaCl	3 ml
DEPC-treated H2O	124.5 ml

ProteinaseK Buffer

• 100 mM Tris pH 8.0
• 50 mM EDTA pH 8.0
• Store 4°C