Question

I need help doing problem number 5 listed below. If someone could show the work required for this problem it would be a great help!

Below is a list of solutions you will need for the mini-prep. Make 5mL of each solution as a team and store each in a 15mL Falcon tube. Store solution 1 and 70% ethanol at 4 degrees Celsius. Solution 3 and Elution Buffer can be stored at room temperature. Show your calculations in the space provided below.

1. Solution 1 (lysis buffer) is (25mM Tris-HCl, pH 8.0, 50mM glucose, 10mM EDTA)

✓ Recall that you have Tris and EDTA stock solutions

✓ You will be provided with a 3M glucose stock solution

2. Solution 2 (Denaturing solution) is (0.2N NaOH, 1.0% SDS)

✓ Do NOT make today (it needs to be made fresh on the day of the mini-prep)

✓ You will be provided stocks of 1N NaOH and 10% SDS

3. Solution 3 (Renaturing Solution) is 5M potassium acetate.

✓ Add 3mL of 5M potassium acetate to 0.575mL of glacial acetic acid and 1.425mL of water

4. 70% ethanol (v/v)

✓ You will be provided with 100% (200 proof) ethanol

5. Elution Buffer (EB) is (10mM Tris-HCl, pH 8.0)

✓ Recall that you have Tris stock solution

Answer 1

To prepare elution buffer - 10mM Tris-HCl, pH=8 following steps are to be followed:

1) Prepare 1M by dissolving 12.1 gm of tris base (C₄H₁₁NO₃ Purchased from Fischer scientific) to 80 ml of nuclease free water.

2) Adjust the pH to 8 by slowly adding approximately 5-6 mL of concentrated HCl (dropwise). Adding HCl increases the temperature hence affect the pH. Thereafter allow the solution to cool.

3) Adjust the volume to 100mL by using nuclease-free water.

4) Filter tris-HCl with sterile filter.

5) To obtain 10mM Tris-HCl of pH 8, you need to dilute 1M Tris-HCl pH=8 by 1:100 with nuclease free water, for example add 1 mL of 1M Tris-HCl of pH 8 to 99 mL of nuclease-free water.

In case you have stock solution of 1M Tris-HCl pH=8 you can straight away dive into step 5 discussed in the answer.