Serial Dilution Calculation HelpWatch
The lengthy question is:
You have been asked by your supervisor to produce a 1 in 2 dilution series of an expensive reagent. Your supervisor has stressed that this is the only bottle there is in the building. Therefore, you have one attempt at doing this accurately. Your supervisor has asked for a 12-point dilution series, made using a 96-well microtitre plate, starting at 1M and working through 1 in 2 dilutions with final volume 100 µL in each well.
You have been given a solution of 1M of the solution and limitless diluent (water). How are you going to do this?
You start by transferring 200µL of the 1M solution into Well 1 of your microtitre plate row. Then you transfer 100µL of diluent (water) into Wells 2–12. Now you need to calculate the volume to transfer from Well 1 to Well 2, mix by pipetting three times, then from Well 2 to Well 3 and so on until Well 12. You can use the same pipette tip for the entire series. You also need to calculate how much to remove from Well 12 to complete your series, as you have been asked for 100 µL per well.
I have also been asked then to work out the concentration of the solution in each step, starting with Well 1 having 1000mM and then working through what well 2 concentration would etc down to well 12.
I have no idea where to start and tried using the C1V1=C2V2 equation that I thought it would be correct but I confused myself when I was asked to work out the concentration at each stage too! Please help!
This is the method for serial dilutions I have been taught for A-Level biology and I don't know if your university has a different method...
- Start by putting 150µL of the solution into well 1.
- For the first dilution, remove 50µL from well 1 with a pipette and add this to well 2.
- Use the distilled water to increase the liquid volume in well 2 to 150µm; this is your first dilution.
- Continue this process until well 12
- After mixing the solution and water, 50µL will need to be removed from well 12 so the final volume is 100µL.
Using this method should reduce the concentration by an order of 10 each time.
To check you can use this formula rearranged:
- volume of stock solution = conc required/conc given x volume required
I hope this helps and good luck