How do you know that you have to make 1: 100 dilution and how is the orginal sto
ID: 543907 • Letter: H
Question
How do you know that you have to make 1: 100 dilution and how is the orginal stock 250 micrograms/mL? Why do you need to add 10 microliters to 990 microliters? Please explain step by step.
7. You are going to use the Bradford Dye Binding Assay to determine the concentration of your protein. But first, you need to set up a standard curve using bovine serum album (BSA). Given the following material listed below, prepare 1.0 mL each of 2.5 g/mL BSA, 5.0 g/mL BSA, 7.5 /mL, 10.0/mL, and 12.5 /mL BSA in water. Make a 100 alution of original tock.5I List of Material Available to You 0.5 mL of 25.0 mg/mL BSA 50 mL of distilled water Twenty-five 5.0 mL test tubea One 200-2000 mL pipetmanv-aso, Deo·p..L One 20-200 mL pipetman One 2-20 mL pipetman All the appropriate pipet tips you need. as,oooExplanation / Answer
Ans. Given, standard BSA solution = 25.0 mg/ mL = 25000 ug/ mL ; [1 mg = 103 ug]
Final volume to be made in each case = 1.0 mL ; [as indicated by “10 uL standard mixed with 990 uL distilled water”]
#1. Preparation of aliquot 1 – 1.0 mL of 2.5 ug/ mL
Using- C1V1 (BSA stock solution) = C2V2 (Aliquot 1)
25000 ug mL-1 x V1 = 2.5 ug mL-1 x 1.0 mL
Or, V1 = (2.5 ug mL-1 x 1.0 mL) / 25000 ug mL-1 = 0.0001 mL
Hence, V1 = 0.1 uL
So, you need to add 0.1 uL BSA stock to 999.9 uL distilled water to make 1.0 mL of 2.5 ug/mL aliquot.
# The volume 0.1 uL is extremely small and may be difficult to measure because we don’t have a pipette to measure this volume. The minimum volume that we can measure is 2.0 uL. So, before preparing the aliquots, we shall prepare the second stock solution (using 1: dilution or as shown below) the concentration of which fulfills the following requirements-
I. During dilution of stock, the first aliquot (the blank) has no BSA stock solution but 1.0 mL water alone. The last aliquot (with maximum concentration) generally has 1.0 mL BSA stock but no water.
So, the concentration of second BSA stock shall preferably be equal to 12.5ug/mL.
II. You can also make the second aliquot with a BSA concentration greater than 12.5 ug/ mL if you want. (like 1:100 dilution as second BSA stock).
III. Since the lowest capacity of micropipette is 2.0 uL, none of the volumes to be pipetted shall be lower than 2.0 uL (you may need to try it few tims to best suit the concentration of second stock solution).
# Preparation of second BSA stock: As calculated above, its shown that 25.0 mg/mL is extremely concentration for preparing the aliquots.
So, we prepare the second aliquot with concentration of 12.5 ug/mL. The volume of second stock shall be 5.0 mL (so that all the aliquots are made from it) and because the maximum volume available to us is the 5.0 mL test tube.
So, we have to prepare the second stock – 5.00 mL of 12.5 ug/mL BSA
Again, using C1V1 (BSA stock solution) = C2V2 (Second aliquot)
Or, 25000 ug mL-1 x V1 = 12.5 ug mL-1 x 5.0 mL
Or, V1 = (12.5 ug mL-1 x 5.0 mL) / 25000 ug mL-1
Or, V1 = 0.0025 mL
Hence, V1 = 0.0025 mL = 2.50 uL
Preparation: Take 2.50 uL of original BSA stock in a clean 5.0 mL tube. Add necessary water to make the final volume to 5.0 mL. The second stock prepared in this step is 12.5 ug/ mL BSA.
# Preparation of 1st aliquot:
Prepare all the aliquots using the second stock solution as shown.
Using C1V1 (second stock solution) = C2V2 (Aliquot 1)
12.5 ug mL-1 x V1 = 2.5 ug mL-1 x 1.0 mL
Or, V1 = (2.5 ug mL-1 x 1.0 mL) / 12.5 ug mL-1
Or, V1 = 0.2 mL = 200.0 uL
Preparation of aliquot 1: Transfer 200.0 uL of second BA stock to a test tube labelled as aliquot 1. Make the final volume to 1000.0 uL with distilled water. It is the first aliquot- 1.0 mL of 2.5 ug/ mL.
Similarly prepare rest of the aliquots using second BSA stock solution.
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