Dilution Practice 1 Scenario: The stock concentration of a mystery substance (MS) is 5 M. Calculate how to dilute to 100 µM (0.1 mM):Use sequential 1:499 and 1:99 dilution steps for accurate preparation.Step 1: Dilute 5 M (5,000,000 µM) to 10,000 µM (500x dilution). 5 M = 5,000,000 µM Target = 100 µM
Scenario: The stock concentration of a mystery substance (MS) is 5 M. Calculate how to dilute to 100 µM (0.1 mM):Use sequential 1:499 and 1:99 dilution steps for accurate preparation.Step 1: Dilute 5 M (5,000,000 µM) to 10,000 µM (500x dilution).
5 M = 5,000,000 µM Target = 100 µM
Overall dilution = 5,000,000/100 = 50,000x
Using sequential steps. 1:499 dilution = 1/500 1:99 dilution = 1/100
Total = 500 x 100 = 50,000
If 1000 µl is total, add 2µl of 5M stock and add 998 µl dH2O
5,000,000 µM x 1/500 = 10,000 µM
Step 2: Dilute 10,000 µM to 100 µM (100x dilution).
If 1000 µl is total, add 10 µl of the 10,000 µM and add 990 µl dH2O.
10,000 µM x 1/100 = 100 µM
The stock concentration of a mystery substance (MS) is 5 M.If the molar mass of MS is 532 g/mol, what’s the concentration of the stock concentration in g/mL?
5 ml/l x 532 g/mol = 2,660 g/l = 2.66 g/ml
To make your life easier, you can use one of many online calculators. You will perform a serial dilution to get 100 uM of MS. Devise a plan to dilute a 5 M MS solution to 100 uM. How many dilution steps will we need? Which tubes should we use? Which pipettes?
Tube 1 In a 1.5 ml microcentrifuge tybe = 998 µl dH2O Add 2 µl of 5M stock Mix well
Tube 2 In a new 1.5 ml tube = 990 µl dH2O Add 10 µl from Tube 1 Mix well
Number of dilution steps = 2
Fill out the following chart to prepare a final reaction with 60 uL reaction volume. Why did we make 100 uM MS if we actually need 40 uM MS? Why not prepare 40 uM in serial dilutions?
C1V1 = C2V2
Loading dye - 6X - 1X 10 µl V = (1X/6X) x 60 µl = 10 µl
MS - 100 µM - 40 µM in 60 µl V = (40/100) x 60 µl = 24 µl
dH2O to 60 µl total 60 - (10+24) = 26 µl
100 µM is a convenient working stock. Because,
