Oneill Lab:WMISH Chemicals

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

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Contents

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
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