Overview
- Tris-EDTA (pH 8.0) solution is used for dissolving and storing DNA.
- Storing DNA in a slightly alkaline condition reduces the risk of depurination.
- Tris functions as a buffering agent and maintains the correct pH of the solution.
- EDTA protects nucleic acid (DNA and RNA) from the action of DNA modifying enzymes and DNases by sequestering divalent cations. In absence of divalent cations, these enzymes are unable to act.
REQUIREMENTS
Reagents and solutions
1M Tris-HCl pH 8.0
0.5 M EDTA pH 8.0
Equipment and disposables
Measuring cylinder
Conical flask / Beaker
Magnetic stirrer with heat control (optional)
Composition of 10X Tris-EDTA solution
100 mM Tris
10 mM EDTA
Composition of 1X Tris-EDTA solution
10 Tris
1 mM EDTA
Note: To prepare 1X solution, take 1 volume of 10X solution, add 9 volumes of deionized / Milli-Q water. Mix it.
Objective
Preparation of 100 ml of 10X Tris-EDTA solution
PROCEDURE
Step 1: Take 88 ml deionized / Milli-Q water in a 250 ml beaker/conical flask. Add 10 ml of 1M Tris.Cl (pH 8.0) and 2 ml of 0.5 M EDTA (pH 8.0). Mix it.
Tips:
◊ One can use manual shaking using a glass pipette to mix the ingredients. Magnetic stirrer makes the mixing process easy and convenient.
◊ It is recommended to add 1M tris and 0.5M EDTA in water instead of adding them together and then adding water.
Step 2: Check the pH. It should be 8.0. If pH is not 8.0, adjust the pH with HCl or NaOH.
Step 3: Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi) from 121-124°C on liquid cycle).
Tips:
◊ You don’t need to autoclave the solution if you have used autoclaved glassware and stock solutions.
◊ If dissolved DNA is to be used for transfection experiments in cell culture, make sure that your Tris-EDTA solution is sterile and free of any live organisms. For this purpose, do all the preparation steps inside the laminar flow hood.
◊ One can also sterilize the solution by filtering through a 0.22 μm filter unit. Filter sterilization removes all suspended particles with a size of more than 0.22 μm which includes most bacteria and their spores but not mycoplasma. Moreover, it does not inactivate enzyme activities (e.g., DNases). Autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorganisms including mycoplasma.
Storage
Both 1x and 10x Tris-EDTA solutions can be stored at 15 – 25 °C (room temperature).
Applications
Tris-EDTA (pH 8.0) is often used to dissolve and store DNA.
| To prepare 10X Trypsin-EDTA of various volumes 10 ml, 50 ml, 100 ml, 500, and 1000 ml), follow the table. | |||||
| Reagents / Volume | 10 ml | 50 ml | 100 ml | 500 ml | 1000 ml |
| 1M Tris, pH 8.0 | 1 ml | 5 ml | 10 ml | 50 ml | 100 ml |
| 0.5 M EDTA, pH 8.0 | 0.5 ml | 1 ml | 2 ml | 10 ml | 20 ml |
| Deionized / Milli-Q water | Adjust to 10 ml | Adjust to 50 ml | Adjust to 100 ml | Adjust to 500 ml | Adjust to 1000 ml |
