I have a transfection experiment to be done.
The stock miRNA is 5nMol. The manufacturer recommends adding 75uL of sterile water, giving me a concentration of 66.67 uM. How can I have a final concentration of 30nM when adding the transfection mix to the wells?
Cells are plated in 2mL
Transfection mix will be around 500uL
If your final Vol is 2,5 ml you need
x µL = (2500µL x 30 mM / 66670 nM) = 1.125µL/transfection (well)
(Ca x Va = Ce x Ve)
If your final vol. is 500µl you need 0.225µL/transfection (well)
x µL = (500µL x 30 mM / 66670 nM) = 0.225µL/transfection (well)
If your final vol. is 2 ml you need 0.9 µL/transfection (well)
x µL = (2000µL x 30 mM / 66670 nM) = 0.9 µL/transfection (well)
If your final Vol is 2,5 ml you need
x µL = (2500µL x 30 mM / 66670 nM) = 1.125µL/transfection (well)
final Vol is 2,5 ml needed
x µL = (2500µL x 30 mM / 66670 nM) = 1.125µL/transfection (well)
https://www.graphpad.com/quickcalcs/Molarityform.cfm
has a calculator for diluting stock solutions
The product data sheet indicates the vial should be diluted in 75uL in order to have an optimum concentration of 66.67uM. I am planning to do some optimization using final concentrations (in final 2.25 mL per well) of 1nM, 3nM, 5nM, 30nM, 50nM.
If I am calculating this correctly, the pipetting amounts for the desired concentrations would be the following:
1: 66,667 dilution to get to 1nM à0.03374
1: 22,223 dilution to get to 3nM à0.10124
1:13,334 dilution to get to 5nM à0.16874
1: 6,667 dilution to get to 10nM à0.33748
1: 2,222 dilution to get to 30nM à1.0126
1: 1,333 dilution to get to 50nM à1.6879
As you can see for some concentrations the pipetting amount is very very low. What do you suggest I should do in order to make it easier to pipette and reduce errors?
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