A hard copy of the answers to these questions is due at the beginning of our next class. Each answer is worth four points. Partial credit may be given if you show your work. One of the lysis buffers I use in my lab requires the following ingredients. Reagent | Mol. Weight | Concentration of stock solution | Volume of stock solution Tris-HCl pH 7.5 | 121.14 g/mol | 1 M | 500 ml HEPES-KOH pH 7.9 | 238.3 g/mol | 1 M | 500 ml MgSO4 | 120.37 g/mol | 1 M | 250 ml (NH4)2SO4 | 132.14 g/mol | 2 M | 250 ml KOAc | 98.15 g/mol | 5 M | 250 ml EGTA pH 7.9 | 380.35 | 0.5 M | 150 ml DTT | 154.25 g/mol | 0.1 M | 5 ml Pepstatin A | 685.89 g/mol | 1 mg/ml | 15 ml ABSF | 239.5 g/mol | 100 mM | 4 ml 1 β 9. How much of each of the above reagents do you have to use to make up the stock solutions with the given volumes? 10. You have a vial containing 100 mg of ABSF. Its molecular weight is 239.5 g/mol. How much water do you need to add to the vial to make a 100 mM stock solution of ABSF? 11. NP-40 is a liquid at room temperature. How much 100% NP-40 do you need to use to make 50 ml of a 10% NP-40 stock solution? 12 β 21. You want to make 2 ml of lysis buffer. How much of each of the stock solutions you made in problems 1 β 10 do you need to add? Reagent | Concentration of stock solution | Final concentration in lysis buffer Tris-HCl pH 7.5 | 1 M | 50 mM HEPES-KOH pH 7.9 | 1 M | 50 mM MgSO4 | 1 M | 10 mM (NH4)2SO4 | 2 M | 100 mM KOAc | 5 M | 12.5 mM EGTA pH 7.9 | 0.5 M | 5 mM DTT | 0.1 M | 2.5 mM Pepstatin A | 1 mg/ml | 1 ?g/ml ABSF | 100 mM | 0.4 mM NP-40 | 10% | 0.01% 22. You also need to add complete protease inhibitor (CPI) to the lysis buffer. Your stock solution concentration is 50x. The final concentration in 2 ml of buffer is 1x. How much do you need to add to your lysis buffer?
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