Question

(Show formula and calculations)

Determine the masses and volumes of each component needed to prepare 5 liters of 100X TAE DNA electrophoresis buffer (10M tris, 20 mM EDTA,)

Tris(M.W)=121.13 g/mol

EDTA (M.W)=292.25 g/ml

Acetic Acid (volume)=114.2 ml

Answer 1

Ans. 1X TAE buffer has following composition: 40 mM Tris, 20 mM acetate and 1 mM EDTA.

# In a 100X TAE buffer solution, the concentration are as follow-

            [TAE] = 100 x 40 mM = 4000 mM = 4.0 M

            [EDTA] = 100 x 1 mM = 100 mM = 0.100 M

            [Acetate] = 100 x 20 mM = 2000 mM = 2.0 M

# Given, required volume of buffer = 5L ; required strength = 100X

#a. Required amount of Tris:

Using              C1V1 = C2V2            - equation 1

            Where, C1 = concentration; V1 = Volume           - Stock solution

                        C2 = Concertation;   V2 = Volume           - 5L, 100X solution

Putting the values in above equation-

            20 mM x V1 = 4.0 M x 5.0 L

            Or, V1 = (4.0 M x 5.0 L) / 10.0 M = 2.0 L

Hence, required volume of 10.0 M Tris stock = 2.0 L

Now,

            Required moles of Tris = [Tris] x Volume of buffer in liters

                                                = 4.0 M x 5.0 L                      ; [1M = 1mol/ L]

                                                = 20.0 mol

            Required mass of Tris = Required moles x Molar mass

                                                = 20.0 mol x (121.13 g/mol)

                                                = 2422.60 g

#b. Required amount of EDTA:

Given, Available EDTA stock = 20 mM

            Required [EDTA] in 100X buffer = 100 mM

Since the EDAT stock is less concentrated than desired [EDTA] in 100X buffer, the stock can’t be used to prepare 100X buffer alone.

We would use solid EDTA sample for this solute.

Required moles of EDTA = [EDTA] x Volume of buffer in liters

                                                = 0.1 M x 5.0 L                      ; [1M = 1mol/ L]

                                                = 0.5 mol

            Required mass of EDTA = Required moles x Molar mass

                                                = 0.5 mol x (292.25 g/mol)

                                                = 146.125 g

#c. Required amount of Acetic acid = 114.2 mL

## Preparation of buffer: Take a clean 5.000 L-standard volumetric flask.

Add 2422.60 g Tris (Or, 2.0 L of 10M EDTA stock) and 146.125 g EDTA to a small amount of water taken in the 5L-flask. Dissolve well by adding small amount of water if needed. Add 114.2 mL acetic acid to the flask and mix well. Make the final volume upto the mark with distilled water. The resultant solution is 5L of 100X TAE buffer.