Hello Lauren Farwell

Primer information sent from a company will list the molecular weight of your primer (g/mol) and the amount of nmoles that they sent. For instance, say, if you have 29.4 nmol of primer and you want to make a 100 μM stock solution in TE, then simply multiply the amount of nmoles by a factor of ten and add that many microliters. In this example you will add 294 μLto make 100μM stock.

It is a general practice is to make a 100 μM stock solution with TE and then dilute to a 10-20 μM working solution using Tris pH 8.3 or filtered ddH2O.

As you mentioned, your requirement is 0.5ul of 0.1uM F primer.

If you make a working stock of 10μM solution of F primer, dilute this working stock further by 1:2 to give a diluted working stock of 5 μM.

Use formula C1 x V1 = C2 x V2

C1= Diluted working stock (5μM)

V1 = Volume required to be added from diluted working stock (XμL)

C2 = Concentration of the primer for the reaction (0.1μM)

V2 = Final volume of the PCR mix (25μL)

Therefore,

5μM x XμL = 0.1μM x 25μL

X = 0.5μL

So, you will add 0.5μL of 5μM (diluted working stock) to 25μL PCR reaction to give a F primer concentration of 0.1μM.

Best.

Also, to convert pmol per vol to concentration is straightforward. 10pmol in 25ul is 10/25 = 0.4pmol.ul-1, and since there are 1e6 ul in a liter, that's 4e5pmol.l-1, or 400nM.

To match this using the calculations above, you'd just add 2ul primer rather than 0.5. Or make your stock up at 20uM and add 0.5ul, which amounts to the same thing.

Sure! Here's how you can calculate the amount of primer to add in microliters (μL) as a μM concentration based on the information provided in the paper:

First, let's convert the 10 pmol of each primer to micromoles (μmol) using the molecular weight of the primer:

For example, if the Clado-SYBRG-PF primer has a molecular weight of 6000 g/mol, then:

10 pmol = 10 x 10^-12 mol

= 1 x 10^-11 mol

= 1 x 10^-8 μmol

Next, let's calculate the total volume of the reaction mix:

The paper states that the total volume of the reaction mix is 25 μL.

Now, let's calculate the final concentration of the primer in micromoles per liter (μM):

To do this, we divide the amount of primer in micromoles (Step 1) by the total volume of the reaction mix in liters:

1 x 10^-8 μmol / 0.025 L = 4 x 10^-7 μM

So the final concentration of each primer in the reaction mix is 0.4 μM (i.e., 4 x 10^-7 μM).

Finally, let's calculate how much of the primer to add in microliters (μL) based on the desired final concentration:

Let's say you want a final concentration of 0.1 μM for the F primer. To calculate the amount of primer to add, we can use the following formula:

Volume of primer (μL) = (Final concentration of primer (μM) x Total reaction volume (μL)) / Primer concentration (μM)

Plugging in the values, we get:

Volume of F primer (μL) = (0.1 μM x 25 μL) / 0.4 μM

= 6.25 μL

So you would add 6.25 μL of the F primer to your reaction mix to achieve a final concentration of 0.1 μM.

I hope this helps! Let me know if you have any questions or if anything is unclear.