Oneill Lab:WMISH Chemicals
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Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology
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.
| 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.
| 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.
| 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.
| 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
| 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.
| 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 |
