Crosby:Protocols

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Protocols : Tris-Cl To prepare a 1 M solution, dissolve 121.1 g of Tris base in 800 mL of H2O. Adjust the pH to the desired value by adding concentrated HCl. pH HCL (ml) 7.4 70 7.6 60 8.0 42 Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume of the solution to 1 L with H2O. Dispense into aliquots and sterilize by autoclaving. If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The pH of Tris solutions is temperature-dependent and decreases ~0.03 pH units for each 1°C increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.6 at 5°C, 25°C, and 37°C, respectively. 2,4-D stock (10 mM) 0.0221 g 2,4-dichlorophenoxyacetic acid (2,4-D) 10 mL ethanol (95%) Dissolve 0.0221 g of 2,4-D in 10 mL of 95% ethanol. Store at -20ºC. Ampicillin stock solution Ampicillin stock solution (100 mg/mL) Ampicillin (sodium salt [sodium ampicillin], m.w. = 371.40) Dissolve 1 g of sodium ampicillin in sufficient H2O to make a final volume of 10 mL. If sterilization is required, prewash a 0.45- or 0.22-μm sterile filter by drawing through 50-100 mL of H2O. Then pass the ampicillin solution through the washed filter. Store the ampicillin in aliquots at -20°C for 1 yr (or at 4°C for 3 mo). Lentiviral Production Detailed protocols describing production of infectious transgenic lentivirus can be found at the Torono lab page