Question

Cell lysis buffer for DNA Fragmentation analysis consists of 10mM Tris-HCl, 10mM EDTA, 0.5% Triton-X-100 and water. How would you make 100 ml of this buffer from a 5M Tris-HCl stock solution, 2.5M EDTA stock solution and 100% Triton-X-100?

Answer 1

Ans. C1V1 = C2V2   - equation 1

C1= Concentration, and V1= volume of initial solution 1         ; i.e. stock solution

C2= Concentration, and V2 = Volume of final solution 2         ; i.e. diluted solution

#a. Required volume of Tris-HCl

            C1 = 5 M        ; V1 = ?           ; V2 = 100 mL

            C2 = 10 mM = 10 x 10^-3 M = 0.01 M                                ; [1 M = 10³ mM]

Putting the values in equation 1-

            V1 = (0.01 M x 100 mL) / 5 M = 0.2 mL

#b. Required volume of EDTA

            C1 = 2.5 M     ; V1 = ?           ; V2 = 100 mL

            C2 = 10 mM = 10 x 10^-3 M = 0.01 M                                ; [1 M = 10³ mM]

Putting the values in equation 1-

            V1 = (0.01 M x 100 mL) / 2.5 M = 0.4 mL

#c. Required volume of Triton-X100

            C1 = 100%     ; V1 = ?           ; V2 = 100 mL

            C2 = 0.5%

Putting the values in equation 1-

            V1 = (0.5% x 100 mL) / (100%) = 0.5 mL

Preparation: Add 0.2 mL of 5M Tris-HCl, 0.4 mL of 2.5 M EDTA and 0.5 mL of 100% Triton X100 to a small volume of water taken in a 100.0 mL standard volumetric flask one at a time. Make sure to added next solution after complete dissolution of the previous one. Make the final volume upto mark with distilled water, mix well for homogenous state. It is the desired solution.