Calculating Concentrations for PCR
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a) Primers :
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);
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:
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
b) Nucleotides :
Example:
i) Make up a 2.5 mM stock solution of dNTPs from stock 100 mM individual dNTPs, supplied by Promega:
- FIRST mix equal volumes of each nucleotide (eg: 50 ul): this gives you 200 ul of 25 mM mixed dNTPs (Remember: concn. expressed in EACH dNTP).
- THEN dilute this (or aliquot) 1/10 with WATER - aliquot into 100 ul amounts and freeze.
- DIG-dUTP supplied (by Boehringer Mannheim) at 25 nmol/25ul = 1 umol/ml = 1mM; final concentration of DIG-dUTP must be 1/10th that of other nucleotides, and [DIG-dUTP] + [dTTP] must = [any other dNTP]. Therefore to get a 1 mM dNTP stock one must dilute DIG-dUTP stock 1/10.
- FIRST dilute separate 100 mM dNTP stocks to 10 mM (eg. 5 ul to 50 ul, in water).
- THEN mix equal volumes (eg. 10 ul) of 10 mM dCTP, dGTP and dATP stock, and 9/10ths volume of dTTP (9 ul). Add equal volume (eg. 10 ul) of of 1 mM DIG-dUTP.
- THEN add water to 10 vol (=100 ul; add 51 ul): final concentration each dNTP = 1 mM; final concn DIG-dUTP = 0.1 mM, and of dTTP = 0.9 mM.
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.
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