PRINS Reactions

1. Mix 5 ||L of primer stock solution with 95 ||L of TE buffer or water for a 5 ||M primer working solution.

2. For each slide, mix in a 1.5-mL microtube:

b. 4 |L of each dNTP working solution, a total of 16 |L.

c. 2 |L of primer working solution.

d. 1 |L of 1 mMbiotin-dUTP or 1 |L of 1 mM dig-dUTP.

f. 23 |L of distilled water.

g. 0.5 |L of Taq DNA polymerase (add immediately before starting the PRINS reaction).

3.2.1. Double PRINS

1. Initiate the PRINS reaction by adding the first reaction solution (50 |L) containing a specific chromosome primer and one of the labels onto a slide. Cover the slide with a cover slip and put the slide on the flat block of the thermocycler.

2. Perform annealing and extension steps according to the different primers used for the detection of different chromosome targets (see Note 8).

3. Wash the slide briefly in 1X PBS solution for 2 min after the first PRINS reaction.

4. Add the second reaction solution containing a primer specific for the second chromosome target and the other labeled dUTP on the same slide.

5. Perform the second PRINS reaction as step 2.

6. Wash the slide in washing buffer for 5 min with gentle agitation (see Note 9).

3.2.2. Triple PRINS

1. Perform double PRINS with two different labels for two different chromosome targets as mentioned previously.

2. Following step 5 of Subheading 3.2.1., wash the slide briefly in PBS solution for 2 min and add the third PRINS reaction solution containing a primer specific for the third chromosome target on the same slide. Use a labeled dUTP that is the same as the one used in the first PRINS reaction.

3. Perform the third PRINS reaction under an appropriate condition of annealing and strand elongation according to the primer used (see Note 8).

4. Wash the slide in washing buffer for 5 min with gentle agitation (see Note 9).

5. Drain the washing buffer from the slide and quickly go to the detection steps.

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