Experiment 1: Preparation of Tris- EDTA Buffer (pH 8)

Aim:

Preparation of TE buffer that will be used to store extracted DNA.

Background:

Tris (Tris-(hydroxymethyl)-aminomethane) is probably the most frequently used buffer

substance in biological treatment. The reasons for this are that Tris is comparatively inexpensive,
very freely soluble in water, is inert in many enzymatic systems and has high buffer capacity.

TE buffer is prepared in pH 7.4- 8.0. This buffer has become the standard buffer for the storage
of nucleic acids. It is used in different pH values. It is generally prepared by mixing Tris buffer
stock solution with an EDTA stock solution (0.5M; pH 8.0). The prepared buffer can also be
stored at room temperature following autoclaving. TE stock solutions are prepared in
concentration of 100X to 1X.

DNA is pH sensitive; Tris can maintain a stable pH. It is an effective buffer between pH7 and 9.
Because of its neutral range, Tris is commonly used buffer in biological labs. DNase are
deactivated because of missing bivalent cations (because of the EDTA), pH is slightly basic
therefore DNA is more easily dissolved than in acid environment, and less prone to acid
hydrolysis. EDTA is a chelating agent which chelates cations e.g Magnesium preventing
degradation by nucleases. Nucleases require bivalent cation for their activity.

Materials:

 Tris-Cl
 EDTA
 1N NaOH
 Distilled water

Part 1: Making 5M Tris-Cl 0.5M EDTA (pH 8.0)

1. Calculate the appropriate amount for making 5M Tris-Cl 0.5M EDTA solution

2. Dissolve in distilled water and make pH 8.0 using NaOH

3. Dilute this 5000X solution into 10X solution with c1v1=c2v2 reaction
4. Now you will get 10X TE appropriate for DNA storage

Alternatively, (Part 2)

1. Make 10 mL 1M Tris-Cl (pH 8.0)

2. Make 10 mL 0.5M EDTA (pH 8.0)

3. To make 10X TE buffer the final concentration needs to be 10mM TrisCl and 1mM EDTA.

4. So to make this concentration of 100 mL 10X TE add 1 mL 1M Tris-Cl (pH 8.0) and 0.2 mL
0.5M EDTA (pH 8.0) in a bottle

5. Now top up the solution to 100 mL by adding 98.8 mL of distilled water.

6. Autoclave the solution to sterilize on liquid cycle (20 min at 15 psi)

7. Store the TE buffer at room temperature (15 to 25 ℃)

Give these a thought:

1. Why we made a higher concentration of TE?

2. Which one is easier to convert, from higher concentration to lower or lower concentration
to higher?
3. Which part was easier, when we made the Tris-Cl and EDTA solution separately and
mixed them later (part 2) or when we made the Tris-EDTA solution together (part 1)?
4. You might have noticed that adjusting the pH was tougher when in was close to target pH
of 8.0. What can be the reason behind that?
5. What do you mean by 10X, 100X, 5000X etc.?