To detect the DNA band in your gel, you need to add ethidium bromide (EtBr) from
ID: 3035469 • Letter: T
Question
To detect the DNA band in your gel, you need to add ethidium bromide (EtBr) from a concentrated stock solution (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 he appropriate volume of concentrated EtBr, you will increase the fila volume of 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?
The answer is not 6.25 ml
b. WHat is the new volume of the agarose solution?
The answer is not 56.25 ml
c. What is the resulting concentration of agarose after you've added the EtBr?
Explanation / Answer
Concertation of stock solution = 40 µg/ mL
Final (desired) concentration = 5 µg/ mL
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.
B: New volume of agarose solution = Initial volume + volume of EtBr stock soln. added
= 50 mL + 6.25 mL = 56.25 mL
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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