You wish to make 1L of sterile lysis buffer soln. that is 10mM Tris-EDTA (pH 8) and 2% SDS. You have plenty of the following stock solns.: 2M Tris-EDTA (pH 8) and 20% SDS. However, SDS cannot be autoclaved and must be added to the soln. after autoclaving is done. Assume that the diluent is dI water.
To prepare 1L of sterile lysis buffer solution, 10mM Tris-EDTA (pH 8) and 2% SDS, follow these steps:
1. Calculate the required volumes of each stock solution:
Since you need a final volume of 1L, it's important to calculate the amount of each stock solution required using their respective concentrations.
a) Tris-EDTA (pH 8):
The final concentration required is 10mM (millimolar). Given that you have a 2M (molar) stock solution of Tris-EDTA (pH 8), you can calculate the volume needed using the formula:
Volume required = (Desired concentration * Desired volume) / Stock concentration
In this case:
Volume required = (10mM * 1L) / 2M = 5 mL
So, you will need 5 mL of the 2M Tris-EDTA (pH 8) stock solution.
b) SDS:
The final concentration required is 2%. Given that you have a 20% stock solution of SDS, you can calculate the volume needed using the formula:
Volume required = (Desired concentration * Desired volume) / Stock concentration
In this case:
Volume required = (2% * 1L) / 20% = 100 mL
So, you will need 100 mL of the 20% SDS stock solution.
2. Autoclave the lysis buffer without adding SDS:
Prepare a solution containing the required 5 mL of 2M Tris-EDTA (pH 8). Add this solution to a beaker with deionized (dI) water, ensuring that the total volume reaches approximately 900 mL. Autoclave this solution without adding SDS, following standard autoclaving procedures for sterilization.
3. Add SDS after autoclaving:
After sterilization, allow the lysis buffer solution to cool to room temperature. Then, add the previously calculated 100 mL of 20% SDS stock solution. Mix gently to ensure it is evenly distributed throughout the solution. This step should be done after autoclaving because SDS cannot withstand high temperatures and can denature during the autoclaving process.
4. Adjust the final volume:
Finally, add deionized water to the solution until the total volume reaches 1L. Mix again gently to ensure homogeneity.
You now have a sterile lysis buffer solution containing 10mM Tris-EDTA (pH 8) and 2% SDS.
To make 1L of sterile lysis buffer solution with 10mM Tris-EDTA (pH 8) and 2% SDS, you can follow these steps:
1. Calculate the volumes of the stock solutions needed.
- We need a final volume of 1L, so we will start with that.
- For 10mM Tris-EDTA (pH 8), we have a 2M stock solution. So, we need to add 10mM Tris-EDTA to the final volume.
- The final concentration required is 10mM.
- The volume of the 2M Tris-EDTA stock solution needed can be calculated using the formula:
Volume of 2M Tris-EDTA = (C1 * V1) / C2
Where C1 is the final concentration required (10mM), V1 is the final volume (1L), and C2 is the concentration of the stock solution (2M).
- For 2% SDS, we have a 20% stock solution. However, SDS cannot be autoclaved, so we need to add it after autoclaving is done.
- The volume of the 20% SDS stock solution needed can also be calculated using the formula above, with the desired concentration of 2%.
- We will calculate this volume later.
2. Autoclave the 1L of diluent water.
- Autoclave a volume of at least 1L of dI water. Make sure the water is properly sterilized.
3. After the autoclaving is done, cool the water to room temperature.
- Allow the water to cool down to room temperature. It is best to work with sterile solutions at room temperature.
4. Add the calculated volume of 2M Tris-EDTA stock solution to the cooled water.
- Following the calculation from step 1, measure and add the appropriate volume of 2M Tris-EDTA stock solution to the cooled water.
5. Calculate the volume of 20% SDS stock solution needed.
- Calculate the volume of 20% SDS stock solution required by multiplying the final volume (1L) by the desired percentage (2%).
- Volume of 20% SDS stock solution = (desired percentage / stock percentage) * final volume
- In this case, the volume of 20% SDS stock solution = (2 / 20) * 1L.
6. Add the calculated volume of 20% SDS stock solution to the lysis buffer.
- After calculating the volume in step 5, measure and add the appropriate volume of 20% SDS stock solution to the lysis buffer.
7. Mix the solution thoroughly.
- Mix the lysis buffer solution by gentle swirling or using a magnetic stirrer until all components are evenly distributed.
8. The resulting solution is now 1L of sterile lysis buffer with 10mM Tris-EDTA (pH 8) and 2% SDS.
- Ensure the solution is properly labeled and stored according to your lab's protocols.
Remember to wear appropriate personal protective equipment (PPE) and follow your lab's safety guidelines while preparing the solution.