Hi,

I used normal qPCR master mix:

- 9 μl 2x SYBR Green PCR MM

- 0.5-5 ng (converted) DNA

- 0.2 μl x2 100 μM Primers

- water up to 18 μl

Worked fine for me!

Best,

Michail

Dear Michail,

Thank you for your response. The primer concentration I received from the Company is of 100 pM. Owing this, which amount do you recommend me to add in my reactions?

Bests,

Hi,

100 pM is really low actually. (In most cases we use 100 μM Primer solutions. I also heard that, for example, Illumina uses 10 μM Primers in Nextera kits)

Maybe you mean that you have 100 pM / litre? For example, when I order primers at Sigma, I select "0.05 μM" which is per litre. When they deliver the primers (they are delivered dry), they mention the volume it should be eluted in to get 100 μM concentration.

For example (one of my orders):

Length - 21

Conc (per litre) - 49,3 nM

MW - 6468

m - 319,1 μg

Volume to get 100 μM = 493 μl water.

Maybe you have the same situation? Then you just need to elute your primer in appropriate volume (you can calculate it based on the MW and m).

Best,

Michail

My primer sheet says that the concentration is100 pM/ μL. So I think I have the same amount as 100 μM /Liter, yes?

That's right!

0.2 μl of each primer per 18 μl of reaction should work fine.

Best,

Michail