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