You are given 10 µL of a DNA stock solution that has 1.0 µg target DNA/µL. The target DNA molecule is 2.9 kb in length.
Describe a set of serial dilutions that would yield a standard curve with the following concentrations:
2 x10^7 copies/µL, 2x10^6 copies/µL, 2x 10^5 copies/µL, and 2x10^4 copies/µL. Make all dilutions in water and do NOT pipet less than 10 µL. You may use as many tubes as you want for making for making dilutions.
To create a serial dilution series with the desired concentrations, we can start with the provided DNA stock solution and perform several dilution steps. Each dilution step will reduce the concentration of the target DNA by a known factor.
Let's begin with the given information:
- DNA stock solution concentration: 1.0 µg/µL
- Target DNA length: 2.9 kb
First, calculate the number of target DNA molecules in the stock solution using the given concentration and the DNA length:
Number of target DNA molecules in stock solution = Concentration (µg/µL) x Volume (µL) x Avogadro's number / Molecular weight (g/mol)
Number of target DNA molecules in stock solution = 1.0 µg/µL x 10 µL x 6.022 x 10^23 molecules/mol / (2.9 x 10^3 g/mol)
Number of target DNA molecules in stock solution = 2.07 x 10^16 molecules
Now, let's calculate the dilution factor required to achieve the desired concentrations for the standard curve:
Dilution factor = Initial concentration / Final concentration
To obtain a concentration of 2 x 10^7 copies/µL:
Dilution factor = 2.07 x 10^16 molecules / 2 x 10^7 molecules
Dilution factor = 1035
To obtain a concentration of 2 x 10^6 copies/µL:
Dilution factor = 2.07 x 10^16 molecules / 2 x 10^6 molecules
Dilution factor = 10,350
To obtain a concentration of 2 x 10^5 copies/µL:
Dilution factor = 2.07 x 10^16 molecules / 2 x 10^5 molecules
Dilution factor = 103,500
To obtain a concentration of 2 x 10^4 copies/µL:
Dilution factor = 2.07 x 10^16 molecules / 2 x 10^4 molecules
Dilution factor = 1,035,000
Now that we have the dilution factors, we can perform the serial dilutions. We need to choose dilution volumes that allow us to pipet at least 10 µL while maintaining sufficient accuracy. Here's one possible set of serial dilutions:
1. For 2 x 10^7 copies/µL:
- Take 10 µL of the stock solution and add it to 10 mL of water.
2. For 2 x 10^6 copies/µL:
- Take 10 µL from the previous dilution and add it to 10 mL of water.
3. For 2 x 10^5 copies/µL:
- Take 10 µL from the previous dilution and add it to 10 mL of water.
4. For 2 x 10^4 copies/µL:
- Take 10 µL from the previous dilution and add it to 10 mL of water.
By following this serial dilution process, you will obtain the desired standard curve concentrations. Remember to thoroughly mix each dilution after adding the components.