What volume of the concentrated EtBr must you add to the 40mL solution of agaros

Question

What volume of the concentrated EtBr must you add to the 40mL solution of agarose to end up with your desired final concentration of 5 mg/mL EtBr?

To detect the DNA bands in your gel, you need to add ethidium bromide (EtBr) from a concentrated stock solution (70 mg/mL) to get a final concentration of 5mg/mL in your agarose solution. Unfortunately, you made your agarose solution (0.8% agarose and 40mL volume) before adding the EtBr. Thus, when you add the appropriate volume of concentrated EtBr, you will increase the final volume of the agarose solution.

What volume of the concentrated EtBr must you add to the 40mL solution of agarose to end up with your desired final concentration of 5mg/mL EtBr? What is the new volume of the agarose solution and what is the resulting concentration of agarose after you've added the EtBr?

Explanation / Answer

Hi,

The concentration of stock solution of ethidium bromide usually(I use for experiments in my lab) is 70 mg/mL

therefore, for making 5mg/mL in 40 mL agarose = 5X 40/ 70 = 2.85 mL

Therefore, 2.85 mL of concentrated EtBr needs to be added to make 40mL solution of agarose to end up with your desired final concentration of 5mg/mL EtBr.

The new volume of agarose solution thus becomes 42.5 mL (40+2.5 mL of EtBr). The concentration of agarose will be 40/42.5= 0.94 times original concentration = 0.94 X 0.8 % = 0.75%.

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