You are planning to digest human genomic DNA with the EcoRI restriction enzyme but realize you do not have any 10X buffer available to add to the reaction. The components of the 10X buffer are: 50 mM NaCl, 100 mM Tris, 10 mM MgCl2, and 0.025 % TritonX-100. You search around the lab and find the following solutions: 5 M NaCl, 1 M Tris, 0.5 % w/v TritonX-100. For MgCl2, you can only find it in powder form. The molecular weight for MgCl2 it is 203.30. Calculate how much of each stock solution, the amount of MgCl2 powder, and how much H2O you would use to make up 100 ml of a 10X solution (assume the MgCl2 powder does not alter the volume). Your answer should clearly indicate how you derived each number. State your volumes in milliliters and milligrams
1 ml
10 ml
5 ml
To calculate the amount of each stock solution, MgCl2 powder, and water needed to make up 100 ml of a 10X solution, we need to determine the molar amounts of each component in the 10X buffer.
1. Start by calculating the molar amounts of each component in the desired 10X solution:
- 50 mM NaCl:
- 50 mM = 50 × 10^(-3) moles in 1 liter (1000 ml) of solution.
- Therefore, the molar amount of NaCl required is 50 × 10^(-3) moles.
- 100 mM Tris:
- 100 mM = 100 × 10^(-3) moles in 1 liter (1000 ml) of solution.
- Therefore, the molar amount of Tris required is 100 × 10^(-3) moles.
- 10 mM MgCl2:
- 10 mM = 10 × 10^(-3) moles in 1 liter (1000 ml) of solution.
- Therefore, the molar amount of MgCl2 required is 10 × 10^(-3) moles.
- 0.025 % TritonX-100:
- 0.025 % = 0.025 g in 100 ml of solution.
- To convert grams to moles, we need to know the molar mass of TritonX-100. However, the molar mass is not provided. Please provide the molar mass of TritonX-100.
2. Once we have determined the molar amounts of each component, we can then calculate the required volumes and weights of the stock solutions and MgCl2 powder:
- 5 M NaCl:
- To obtain 50 × 10^(-3) moles of NaCl, we need to calculate the volume of the 5 M NaCl solution required.
- Moles = Molarity × Volume (in liters).
- Volume (in liters) = Moles / Molarity.
- Moles of NaCl = 50 × 10^(-3) moles.
- Molarity of the 5 M NaCl solution = 5 mol/L.
- Volume (in liters) = (50 × 10^(-3)) / 5 = 0.01 L.
- Volume (in milliliters) = 0.01 L × 1000 = 10 ml.
Therefore, you would use 10 ml of the 5 M NaCl solution.
- 1 M Tris:
- To obtain 100 × 10^(-3) moles of Tris, we need to calculate the volume of the 1 M Tris solution required.
- Moles = Molarity × Volume (in liters).
- Volume (in liters) = Moles / Molarity.
- Moles of Tris = 100 × 10^(-3) moles.
- Molarity of the 1 M Tris solution = 1 mol/L.
- Volume (in liters) = (100 × 10^(-3)) / 1 = 0.1 L.
- Volume (in milliliters) = 0.1 L × 1000 = 100 ml.
Therefore, you would use 100 ml of the 1 M Tris solution.
- 0.5 % w/v TritonX-100:
- To obtain 0.025 g of TritonX-100, we need to calculate the volume of the 0.5 % w/v TritonX-100 solution required.
- 0.025 g = 0.025/100 g in 1 ml of solution (since the concentration is in w/v %).
- Therefore, you would use 0.025 ml of the 0.5 % w/v TritonX-100 solution.
- MgCl2 powder:
- The amount of MgCl2 powder required can be calculated using its molar mass.
- Molar mass of MgCl2 = 203.30 g/mol (given).
- Moles = Mass/ Molar mass.
- Moles of MgCl2 = (10 × 10^(-3) moles) = 10 × 10^(-3) mol.
- Mass = Moles × Molar mass.
- Mass = 10 × 10^(-3) mol × 203.30 g/mol = 2.033 g.
Therefore, you would use approximately 2.033 g of MgCl2 powder.
3. Finally, we can calculate the volume of water needed to make up the total volume of 100 ml:
- Total volume = 50 ml (NaCl) + 100 ml (Tris) + 0.025 ml (TritonX-100) + 100 ml (water) = 250.025 ml.
- Volume of water = Total volume - Sum of other solutions' volumes.
- Volume of water = 100 ml - 250.025 ml = -150.025 ml (negative value due to rounding, indicating that the desired volume cannot be achieved with the given stock solutions).
Therefore, the required amount of water cannot be determined based on the given information.
In summary, to make up 100 ml of a 10X solution for digesting human genomic DNA with the EcoRI restriction enzyme using the available stock solutions, you would use 10 ml of the 5 M NaCl solution, 100 ml of the 1 M Tris solution, 0.025 ml of the 0.5 % w/v TritonX-100 solution, and approximately 2.033 g of MgCl2 powder. However, the volume of water cannot be determined with the information provided.