Primer Resuspensions and Dilutions:
---Resuspension of newly arrived primers:
First, spin all tubes to ensure that any dried pellets are not trapped in the caps.
Dissolve fresh primer with the 10X volume of TE as its concentration:
For example, if tube contains 25 nmoles of primer add 250 ul of TE.
This will give you a concentration of 0.0001 M or 0.1 mM
(25 nmoles/L) (X moles/250 x 10-6 L) = .0001 mole/L = 0.0001 M = 0.1 mM = 100 µM
This is your ‘stock’ primer.
---Dilution of prepare ‘working’ primer solution for use:
Dilute this stock 1:10, to give a concentration of 10 M
For example, if you want 200 L of a 10 M primer, you would dilute 20 L of stock
primer in 180 L of water or TLE.
C1 x V1 = C2 x V2
(100 M * X L) = (10 M * 200 L), solve for X
X = (10 M * 200 L)/100 M
X = 20 L of primer stock dissolved in 180 L of water or TLE for a final volume of 200
L.
---From this, use ~0.5-1 l in a typical PCR reaction. This will give you a final
concentration of 5-10 pmoles in a PCR reaction.
(Because 1 L of 10 M = 10 moles/10-6 L = 0.00001 mole/l = 10 pmol/l)