Microscopy Buffer (1X)
(pH 7.3; ~320mOsm/kg)
| Component | Final Conc. |
|---|---|
| NaCl | 130.00mM |
| KCl | 5.36mM |
| MgSO4 | 0.80mM |
| Na2HPO4 | 1.00mM |
| Glucose | 25.00mM |
| HEPES | 20.00mM |
| Na-Pyruvate | 1.00mM |
| NaHCO3 | 2.50mM |
| Ascorbic Acid | 1.00mM |
| CaCl2 | 1.50mM |
| BSA | 1.5mg/ml |
Prepare 10x stock solution:
| 1300.00mM NaCl | 76.00g |
| 53.60mM KCl | 4.00g |
| 8.00mM MgSO4, 7H2O | 1.97g |
| Na2HPO4, 2H2O | 1.97g |
| 250.00mM Glucose | 45.00g |
| 200.00mM HEPES | 47.70g |
| 10.00mM Sodium Pyruvate | 1.10g |
| dWater | to 1L |
- Adjust pH to 7.5 with Tris base powder (~9g)
- Filter sterilize and store at 4°C
To make the working buffer:
| Component (in order of addition) |
Add from: | For 100mL | For 250mL | For 500mL |
|---|---|---|---|---|
| Water | - | 85ml | 220ml | 445ml |
| Stock | 10X Stock Powder | 10ml | 25ml | 50ml |
| Ascorbic Acid | Powder | 0.0198g | 0.0495g | 0.099g |
| NaHCO3 | Powder | 0.021g | 0.0525g | 0.105g |
| BSA | Powder | 0.15g | 0.375g | 0.75g |
| CaCl2 | 1M Stock | 0.15ml | 0.375ml | 0.75ml |
- Bring to final volume after all components have been added.
- Adjust to pH 7.3 with 1M Tris (If verification of osmolarity is necessary, adjust pH without BSA, then check osmolarity. Add BSA to solution after measurement.)
- Filter sterilize
To prepare a nominally Ca-free buffer:
- Prepare Working Buffer as above, omitting CaCl2
To prepare buffer with 100nM Ca (pCa=7):
- Prepare Working Buffer as above, omitting CaCl2
- Add 100µM EGTA (20µl of 0.5M EGTA/100ml 1X Buffer)
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