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Calculating Concentrations for PCR

2024-11-19 PCR 加入收藏

a) Primers :i) Oligonucleotide primers are generally supplied as "so many O

a) 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).

b) Nucleotides :

Stocks of nucleotides for PCR (or other procedure) are NEARLY ALWAYS dNTP s (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:

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.

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 :

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: