To detect the DNA bands in your gel, you need to add ethidium bromide (EtBr) fro

Question

To detect the DNA bands in your gel, you need to add ethidium bromide (EtBr) from a concentrated stock soluion (40 ug/ml) to get a final concentration of 5 ug/ml in your agarose solution. Unfortunately, you made your agarose solution (0.8% agarose and 50 ml volume) before adding the EtBr. Thus, when you add the appropriae volume of concenrated EtBr, you will increase the final volume of the agarose solution.

a. What volume of the concentrated EtBr must you add to the 50 ml solution of agarose to end up with your desired final concentration of 5 ug/ml EtBr?

b. What is the new volume of the agarose solution?

c. What is the resulting concentration of agarose after you've added the EtBr?

Explanation / Answer

Ans. Concertation of stock solution = 40 µg/ mL

Final (desired) concentration = 5 µg/ mL

Part A: Total amount of EtBr in 50 mL agarose solution = desired [EtBr] x 50 mL

                                                = (5 µg/ mL) x 50 mL = 250 µg

Volume of stock solution containing 250 µg EtBr = amount of EtBr / [EtBr] stock

                                                = 250 µg / (40 µg/ mL) = 6.25 mL

So, add 6.25 mL of stock EtBr to agarose solution.

Part B: New volume of agarose solution = Initial volume + volume of EtBr stock soln. added

                                                = 50 mL + 6.25 mL = 56.25 mL

Part C: Resultant concentration = (Total mass of EtBr / Total volume) of final solution

                                                = 250 µg / 56.25 mL

                                                = 4.44 µg/ mL

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