Preparation of DNA Duplex for Spotting

A common amino-modified 16-mer is annealed to a 34-mer containing a complementary region and a transcription factor binding site (Fig. 5). The latter is then made double-stranded in a simple extension reaction.

1. In each well of a 96-well plate mix the following: 30 yL of 100 yM specific primer, 15 yL of 100 y M common primer, and 5 yL of 10X NEB3 buffer.

2. Anneal the mixture of two oligonucleotides in a PCR block: 1 cycle at 94°C for 1 min; and 70 cycles at 94°C for 1 min and -1°C per cycle.

3. Transfer a 20-yL aliquot to a 0.6-mL tube and add the following extension mix (see Note 4): 2 yL (10 U) of DNA Polymerase I, Large (Klenow) Fragment, 3 yL of 10X EcoPol or NEB2 buffer, 2 yL of 2.5 mM dNTP mix, and 3 yL of dH2O.

4. Incubate at RT for 30 min and then at 37°C for 15 to 30 min.

5. Add 90 pL of 100% ethanol and 3 pL of 3 M sodium acetate (pH 5.2).

6. Place at -20°C overnight and then precipitate by centrifugation at 16,000g for 30 min in a benchtop centrifuge at 4°C.

7. Aspirate supernatant and add 120 pL of ice-cold 70% ethanol. Wash DNA by spinning at 16,000g for 30 min at 4°C.

8. Aspirate supernatant and allow samples to dry at RT. Resuspend in 30 pL 1X spotting solution. Transfer duplexes to a 384-well plate. Spin plate at 200g. Samples are now ready to be spotted.

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