PRINS Reaction

1. Prepare a reaction mixture in a final volume of 50 pL containing: 0.2 mM of dATP, dCTP, and dGTP; 0.02 mMof dTTP; 0.02 mMof FITC-12-dUTP; 50mM KCl, 10 mM Tris-HCl, pH 8.3; 1.5 mM MgCl2; 0.01% BSA; 200 pmol of oligonucleotide primer; and 2.5 U of Taq DNA polymerase. In practice, mix in a sterile microcentrifuge tube: 1 pL of each 1:10 diluted dATP, dCTP and dGTP; 1 pL of the 1:100 diluted dTTP; 1 pL of FITC-12- dUTP; 1 pL of BSA; 5 pL of 10X Taq buffer, 0.5 pL of the Taq DNA polymerase, 4 pL of the primer specific for chromosome 9, and distilled water to 50 pL.

2. Place the reaction mixture under a 22 x 32 cover slip on the denatured slide and transfer to the heating block of the thermal cycler.

3. Set up the PRINS program for the appropriate temperatures and start the reaction. Two distinct programs can be used:

a. The standard PRINS program consisting of two steps: 5 min at the annealing temperature (56°C for chromosome 9 primer according to Table 1 in Chapter 6) and 5 min at 72°C.

b. The fast PRINS program (according to Subheading 3.2.1., step 3 in Chapter 6) consisting of a unique 5 min step at the specific annealing temperature of the chromosome 9 primer (56°C).

While the reaction is running, prepare the PNA reaction mixture as described

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