如何设计引物（二）

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:

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:

NEED to dilute mix 1/20; therefore use 1.25 ul dNTP labelling mix per 25 ul reaction volume (1/20 = 5/100 = 1.25/25).

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