如何设计引物（一）

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 330.Y daltons

And: X OD/ml = 37.X ug/ml = 37.X mg/l = 37.X /330.Y mM = 37.X.1000/330.Y 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).