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APPENDIX A
Buffer Preparation
Buffer Solvent: De-ionized water (ddH2O)
Buffer Solute Ingredients/ Proportions:
To prepare 200ml of KRB. (prepare right before use)
1. Add 20ml of Solution 1 to 100ml of ddH2O
2. Bubble gently with 95% O2/5% CO2 for 10 minutes
3. Add 20ml of Solution 2
4. Repeat bubbling as above
5. Add 20ml of Solution 3
6. Add 1.2ml (6mM) of 1M HEPES
7. Adjust pH=7.4 with NaOH. After adding the above solutions the pH≈7.08
8. Bring volume to 200ml with ddH2O
9. Add 0.4g (0.2%) of BSA (Sigma Cat. A7888 OR A6003)
10. Filter Sterilize through 0.22μ filter
Notes:
a. When making Solution 2 dissolve CaCl2 first
b. Solution 1 and 2 should be stable for several weeks at 4℃
c. Solution 3 is made fresh every time. Prepared 50ml 10X stock solution dissolving 1g of NaHCO3 in 50ml of
ddH2O
d. Gassing times may be overkill, but this minimizes the risk of precipitate formation
e. The working solution should last for 12-24h; beyond this, there is risk of precipitate formation.
f. KRBH= KRB containing HEPES
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