DNTP, solution preparation

Kinetic substrate solution Prepare as for dNTP substrate solution above, but using dNTP duo solution instead of dNTP trio solution. 

To each set of microfuge tubes set up in step 2, add 5 oL of one of the sets of dNTP inhibitor solutions prepared as described in step 3 and mix. (If required, tubes may also be stored frozen for later use at this stage). 

For first-strand cDNA synthesis SUPERSCRIPT II RT reverse transcriptase (200 U/pL) supplemented with 5X RT buffer (250 mM Tris-HCl, pH 8.3, 375 mM KC1, 15mM MgCl2) (Gibco-BRL), random hexamers (50 ng/pL), 0.1 M DTT, 10 mM dNTP mix, and DEPC-treated water. These solutions should be prepared as RNase-free. We utilize solutions supplemented with cDNA synthesis kit (Gibco-BRL)... 

Prepare stock solutions of reaction buffer (10X RAPD buffer) and deoxynucleotides (10X dNTPs). Larger quantities of both solutions can be prepared, subdivided into aliquots, and stored frozen at —20°. 

X dNTPs, ddNTP mixes are prepared from 10 mM stock solutions of each dNTP (in 10 mM Tris-HCl, pH 7.5) and from 1 mM stock solutions of each ddNTP (in 10 mM Tris-HQ, pH 7.5)... 

Prepare dNTP stock solution of dCTP, dTTP, and dGTP containing 0.1 mM of each. 

PCR Grade 10 ruM dNTP Mix (Invitrogen). Prepare a 2.5-mM working stock solution of dNTPs in sterile milli-Q (or ddH O) water for regular use. Store all PCR reagents at -20°C. 

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