Protocol 111 Clone preparation and array fabrication

1. Prepare a PCR premix containing the following reagents (the amount of each reagent is given for one PCR reaction. They should be scaled up appropriately according to the number of PCRs being set up).

80.5 |l sterile distilled water

1.0 |l Taq 2000 DNA polymerase

Dispense a 98-|l aliquot of the PCR premix into a microtiter plate (or microcentrifuge tube) containing 2 |l of DNA sample (approximately 50-100 ng).

2. Place the microtiter plate in a thermal cycler. Suggested cycling conditions: denaturing at 94°C for 3 min, followed by 30 cycles 94°C for 30 s, 37°C for 30 s and a linear ramp from 37°C to 72°C and 72°C for 1 min, followed by a final extension of 10 min at 72°C.

3. DOP PCR products are purified using a multiscreen plate (Millipore) and finally dissolved in 55 |l TE.

4. Check amplified DNA quality and fragment length by agarose gel electrophoresis on a 1% 0.5 x TBE agarose gel. Load 1-2 |l of the purified DOP PCR product.

5. Dry the amplified BAC clones in a microtiter plate using a speedvac.

6. Dissolve BAC clones in spotting buffer containing 30% DMSO.

7. Arrays can now be prepared for spotting onto aminosilane-coated slides according to manufacturers instructions using a split-pin system. Depending on the number of clones to be spotted, they can be spotted once or in replicate.

8. Dry the slide overnight in the arrayer.

9. Crosslink the slide according to procedures provided by the different manufacturers.

10. Store the slide in an exicator at room temperature for up to 6 months.

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