Question

Describe how would you prepare the GTE (50 mM Glucose, 20 mM Tris, EDTA) Resuspension Solution for use in a mini-prep. You have the following stock solutions: 0.5 M glucose, 0.2 M Tris-HCl (pH 8.0), 0.5 M EDTA (pH 8.0).
How would you prepare 150 mL of GTE re-suspension solution using the above stocks?

Also, describe how to prepare the solution in appropriate container and aseptic technique.

Answer 1

Glucose-tris-EDTA (GTE) buffer is used during DNA extraction to resuspend cells before the cell lysis is carried out. The primary purpose of the GTE buffer is to ensure a homogenous solution of cells from which the DNA has to be extracted. A homogenous solution will ensure complete lysis of the cells while also providing a stable environment for the released DNA. Glucose in the GTE buffer maintains the osmolarity of the solution, tris(hydroxymethyl)aminomethane (tris) maintains the pH and EDTA prevents DNA degradation.

To prepare a working concentration of the GTE buffer, the following formula can be used.

V1 x C1 = V2 x C2

Where,

V1 = volume of stock solution

V2 = final volume of working solution

C1 = concentration of stock solution

C2 = concentration of working solution

We need 150ml of 50 mM Glucose, 20 mM Tris, 10 mM EDTA.

We have the following stock solutions

0.5 M glucose, 0.2 M Tris-HCl (pH 8.0), 0.5 M EDTA (pH 8.0).

1. Preparation of 50 mM glucose

V1 = has to be calculated

V2 = 150ml

C1 = 0.5M = 0.5 x 1000 = 500 mM

C2 = 50 mM

V1 x C1 = V2 x C2

V1 x 500 = 150 x 50

V1 = (150 x 50) / 500 = 7500 / 500 = 15 ml

15 ml of 0.5M glucose

2. Preparation of 20 mM Tris

V1 = has to be calculated

V2 = 150ml

C1 = 0.2M = 0.2 x 1000 = 200 mM

C2 = 20 mM

V1 x C1 = V2 x C2

V1 x 200 = 150 x 20

V1 = (150 x 20) / 200 = 3000 / 200 = 15 ml

15 ml of 0.2M Tris

3. Preparation of 10 mM EDTA

V1 = has to be calculated

V2 = 150ml

C1 = 0.5M = 0.5 x 1000 = 500 mM

C2 = 10 mM

V1 x C1 = V2 x C2

V1 x 500 = 150 x 10

V1 = (150 x 10) / 500 = 1500 / 500 = 3 ml

3 ml of 0.5M EDTA

Preparation of GTE (50 mM Glucose, 20 mM Tris, EDTA) Resuspension Solution

1. Take a sterile 500ml beaker.

2. Using a sterile pipette transfer 15 ml of 0.5M glucose to the beaker.

2. Using a fresh sterile pipette transfer 15 ml of 0.2M Tris to the same beaker.

3. Using a fresh sterile pipette transfer 3 ml of 0.5M EDTA to the same beaker.

4. Measure 117 ml of distilled water in a sterile cylinder and add to the same beaker.

5. Mix well