Volume of Primers - (May/02/2015 )

Please correct me if i am wrong or kindly suggest a way to solve this:

I want to calculate the volume of primer (microliter) that i need to use for PCR reaction:

x = final conc. of primer X total vol. of reaction/ stock conc. of primer

x = 0.3 * 20/10 = 0.6 microliter of primer

Looks fine... it's just a rearrangement of C1V1=C2V2.

bob1 on Sun May 3 20:58:19 2015 said:

Looks fine... it's just a rearrangement of C1V1=C2V2.

Thanks bob, I also thought the same.

Its V1= c2v2/c1

It REALLY helps if you use units with your math. Units cancel, just like numbers, and it makes many mysterious things obvious. I would never have written the equation as you did. Rather:

x ul = 0.3 uM * 20 ul / 10 uM

The uM cancels, and you have x ul = 0.6 ul

If your  units fail to cancel in the right way, your equation is wrong.

No comment on your math, I think the other scientists covered it.

Next time you order primers, or if you have a master stock that you can dilute, take note of the amount of DNA (usually in nanomoles) that the company delivers to you. Calculate the volume of solvent (water or Tris pH 8) to add to make a 100 pmol per ul stock. I typically add 100 pmol of each primer (forward and reverse) per reaction, so I just add 1 ul of each primer to the reaction mix. Less calculation AND you don't have to pipet less than 1 ul.

For example, if the primer company delivers 350 nmol of primer (typical amount), add 3500 ul of buffer to make 0.1 nmol/ul or 100 pmol/ul.