You can first dilute your primer stock (100 uM) to a concentration of 25 uM, by taking out 25 uL of the stock solution and add 75 uL of nuclease-free water to a final volume of 100 uL (this way you also don't contaminate your original primer stock). Then you can add 3 uL of the diluted primer (25 uM) to your 25 uL PCR mix. I hope this helps.

You can first dilute your primer stock (100 uM) to a concentration of 25 uM, by taking out 25 uL of the stock solution and add 75 uL of nuclease-free water to a final volume of 100 uL (this way you also don't contaminate your original primer stock). Then you can add 3 uL of the diluted primer (25 uM) to your 25 uL PCR mix. I hope this helps.

Usually when you recieve your synthesized primers (lyophylized) you'll get a datasheet with all information and also they provide which volume to dissolve in to obtain a 100 µM solution (used by many as stock). You can the dilute your primers to 10 µM using nuclease-free water and store as your stock in the freezer. To prepare 10 µM working solution disolve 10 µL from the stock (100 µM) in 90 µL water, mix and spin down.

You can use the prepared primer's working solution (10 µM) to adjust the primre's concentration in your PCR master mix with the following equation:

C x V=C' x V' ...... where

C= concentration of the primers in the working solution (10 µM)

V= the volume you need (? µL)

C'= concentartion of the primers in the PCR mix

V'= total volume in the PCR reaction mix

Simply, for a 25ul reaction, we would generally start with 1ul of each primer working stock (10 For a 25ul reaction, we would generally start with 1ul of each primer working stock.)

Dear Rion Zaren

I totally agree with Jennifer Tu and would have recommended the same to avoid contamination. It would be easier to calculate for 25/50 µl PCR reaction volume from 25 mM stock.

Best wishes.

https://www.aphl.org/programs/newborn_screening/Documents/2015_Molecular-Workshop/Lab-Math-Primer-Preparation-and-Dilution.pdf

Hi Zaren,

I believe that after reconstituting your primer sets, the conc is 100 uM)

Use the C1V1=C2V2 formular to prepare a stock of 10uM of your primer set in aliquots from the supplied 100uM stock. (i.e add 1 uL from stock to 9 uL of nuclease free water) to make your working stock solution, store tubes at - 20 degrees

For each vial thawed when you want to make your master mix of 25 uL volume, add 7.5uL of your prepared working stock solution to making final conc of 3uM.... This reaction might form primer dimers. I will recommend if you can use a lesser concentrated primer set like 0.3uM. This means you will only add 0.75uL of your prepared primer stock working solution.

If primer dimers forms, you should do a temperature gradient PCR.

I hope this make sense. Thank you

I agree with both Dr. Tu and Dr. Ezechukwu. Remember that 100 uM is the same as 100 pmol/ul, so decide what you want to use as a working stock solution (10 uM or 25 uM). If you're going to start with a 25 uM working stock like Dr. Tu recommended, add 25 uL of 100 uM solution to 75 ul of nuclease-free water. With a working stock at 25 uM, divide 25 uM by your desired 3 uM final concentration. That gives you a dilution factor of ~8.33. Now take your reaction volume (25 ul), divide it by your dilution factor of ~8.33, and you'll find that you need to add ~3 ul of 25 uM primer to your master mix to obtain a final concentration of 3 uM in a 25 ul reaction.

I strongly agree with Jennifer Tu

I agree with both Dr.Tu and Dr, Ezechukwu.

I agree with dr Jennifer_Tu