Question

8) A buffer used to denature proteins incudes the following components: 25 mM Tris-HCI, pH 8.2 . 8 M urea 200 mM glycine · 10 mM β-mercaptoethanol (BME) 250 mM NaCI In the lab you have a 1 M solution of Tris buffer at pH 7.0, a bottle of BME at 14.3 M and solid urea, glycine, and NaCI. How you would prepare this buffer? Provide a step by step description of how to make this buffer with details including rinsing glasses, how you would measure each item, volumes of water added to beakers and so on. Use the back of this page for your answer if necessary. (5 points)

Answer 1

All the components given for making buffer will be made separately first and stored in different bottles then they will be added to make the final concentration. First wash the glasswares with detergent and water. And when they have dried wipe them with 70%alcohol.

For 25mM Tris HCl, here 25mM means 1/40 of a mole per litre since . 1M means 1 mole of a substance per litre. Therefore divide the molecular weight of Tris (121.14) by 40 and weigh this amount. Mix this in with 900ml of filtered or distilled water. Now adjust the pH usin HCl and NaOH. Since you have been given 1M soln you'll have to dilute. 1 M is 1000mM. That means 4x it has to be diluted. To 100 ml of 1M soln add 400ml of distilled water and adjust pH. Dilute similarly BME.

Urea, glycine and NaCl will be made according to their molecular weight. Dissolve, for NaCl 58.44 g in 1000ml of water. It will be one molar or 1 M. But you need only 250mM either you can dilute or add required amount of salt to water. Do the calculations on your own , you'll learn better.