1. Serial Dilutions

2. What are serial dilutions?  Also called a “dilution series”, serial dilutions are, essentially, dilutions of dilutions.

3. Why do we need serial dilutions? This flask contains 10 mg/mL Green Fluorescent Protein (GFP)* *GFP is a protein that glows green under UV light Problem: Make 10 mL of a 1 ng/mL solution of GFP GFP 10 mg/mL Solution: Convert both concentrations to g/mL. 10 mg/mL = 10 -2 g/mL 1 ng/mL = 10 -9 g/mL Use C 1 V 1 = C 2 V 2 (10 -2 g/mL)(y) = (10 -9 g/mL)(10 mL) y = (10 -9 g/mL)(10 mL) (10 -2 g/mL) Y = 10 -6 mL or 0.001  L To make this solution, you would need to add 0.001  L of GFP stock to enough water to make 10 mL total. But we cannot pipet volumes smaller than 1  L accurately!

4. So what do we do? What is the total dilution that we need to make? Stock concentration: 10 mg/mL= 10 -2 g/mL Final concentration: 1 ng/mL = 10 -9 g/mL The overall dilution is: Final10 -9 g/mL = 1 Stock10 -2 g/mL10 7 We need to make a 1/10 7 dilution of our GFP stock.

5. Let’s plan our dilution scheme We can plan a series of dilutions such that the volume of GFP pipetted each time CAN be measured accurately… The key is that the product of our dilutions must equal 1/10 7 GFP 10 mg/mL This tube will be a 1/10 7 dilution of the GFP stock!! Continue….

6. Let’s plan our dilution scheme GFP 10 mg/mL Dilution Plan: Dilution of previous tube: 1/100 1/100 1/100 1/10 Dilution of GFP stock: 1/100 * 1/100 * 1/100 * 1/10 = 1/10 7 Remember that the dilution of stock in the final tube is determined by multiplying the dilutions of each of the tubes in the serial dilution! Continue….

7. Now to the details… Now we know that, to make a final dilution of 1/10 7, we can make the series of dilutions shown. All we need to do is calculate how to make the individual dilutions… Let’s make 10 mL of each dilution… GFP 10 mg/mL Dilution of previous tube: 1/100 1/100 1/100 1/10 Dilution of GFP stock: 1/100 * 1/100 * 1/100 * 1/10 = 1/10 7 0.1 mL GFP stock + 9.9 mL water 0.1 mL tube 1 + 9.9 mL water 12 0.1 mL tube 2 + 9.9 mL water 3 1 mL tube 3 + 9 mL water 4

8. Let’s determine the concentrations: All we need to do is to multiply the dilution made of the previous tube by the concentration of the previous tube… For example: Concentration of tube 1 = 1/100(10 mg/mL)= 0.1 mg/mL, or 10 -4 g/mL GFP 10 mg/mL Dilution of previous tube: 1/100 1/100 1/100 1/10 Dilution of GFP stock: 1/100 * 1/100 * 1/100 * 1/10 = 1/10 7 0.1 mL GFP stock + 9.9 mL water 0.1 mL tube 1 + 9.9 mL water 12 0.1 mL tube 2 + 9.9 mL water 3 1 mL tube 3 + 9 mL water 4 Tube 1: 10 -4 g/mL Tube 2: 10 -6 g/mL Tube 3: 10 -8 g/mL Tube 4: 10 -9 g/mL

9. Done! Remember where we started: Problem: Make 10 mL of a 1 ng/mL solution of GFP Tube 4 contains 10 mL of 10 -9 g/mL GFP, or 1 ng/mL GFP!!!! GFP 10 mg/mL 0.1 mL GFP stock + 9.9 mL water 0.1 mL tube 1 + 9.9 mL water 12 0.1 mL tube 2 + 9.9 mL water 3 1 mL tube 3 + 9 mL water 4 Tube 1: 10 -4 g/mL Tube 2: 10 -6 g/mL Tube 3: 10 -8 g/mL Tube 4: 10 -9 g/mL