Calculating Concentrations for PCR

1. Primers:

i) Oligonucleotide primers are generally supplied as "so many OD units/ml" - but what does this mean, in terms of mg/ml, or mmol/ml, etc?

Given: a primer is Y nucleotides (nt) long;

Given: the MW of ssDNA is (330 daltons per nt) x (length in nt) (Sambrook et al., 1989; p. C.1);

Given: the concentration of primer (=ssDNA) producing an OD of 1 at 254 nm in a 1 cm cuvette, is 37 ug/ml;

Then: the MW of the primer is 330Y daltons

And: X OD/ml = 37X ug/ml = 37X mg/l = 37X /330Y mM = 37X1000/330Y uM

For example:

B 88/77 primer - a 17-mer oligodeoxynucleotide - as supplied is 12.6 OD units/ml. We need to make a 5 uM stock solution for PCR.
MW: 17 x 330 = 5610

Concentration: 12.6 OD x 37 ug/ml = 466 ug/ml = 466 mg/l = 0.466 g/l

Molarity: 0.466/5610 = 0.000083 Molar = 83 uM

Therefore: we need 5 ul of oligo stock solution in 83 ul (+78 ul water) to make a 5 uM solution (if 1 ul in 83 ul gives a 1 uM soln...)
ii) Calculation of amounts for PCR reactions: if we need a final concentration of 0.5 uM oligo in the PCR reaction mix (final volume 50 ul), we add 5 ul of 5 uM stock to the reaction mix (1/10 final dilution).

2. Nucleotides:

Stocks of nucleotides for PCR (or other procedure) are NEARLY ALWAYS dNTPs (deoxynucleotides), and concentrations is almost always given in EACH dNTP: that is, the given concentration is EACH nucleotide in the mix, NOT the total concentration. This means that a 2.5 mM dNTP mix for PCR contains 2.5 mM of EACH dNTP, and 10 mM TOTAL dNTPs.

Example:

i) Make up a 2.5 mM stock solution of dNTPs from stock 100 mM individual dNTPs, supplied by Promega:

ii) Prepare a 1 mM stock of dNTPs with dTTP substituted to 10% (w/w) by digoxigenin-11-dUTP (DIG-dUTP) for use as a labelling mix for PCR labelling of PCR products:

GIVEN:

iii) USE mix made above at 50 uM each dNTP in a PCR reaction mix, final volume 25ul:

To make mastermix: multiply amount of dNTP per reaction by number of reactions.