Screening for lacZ Transgene Expression Membrane Preparation

1. Spot 10 |g and 5 |g of DNA onto a positively charged nylon membrane, and air-dry the membrane thoroughly (10 min).

2. Denature the DNA by floating the membrane DNA side up in buffer 1, then buffer 2, and then buffer 3 each for 5 min.

3. Expose the wet membrane to UV light using the Stratalinker at 12 J/cm2. The Stratalinker calculates the required time to deliver 1200 J automatically. The membrane can now either be used immediately or be air-dried, sealed in plastic and stored at 4°C until required.

4. Wet the membrane in 5X SSC (1 min), and incubate the membrane in prehybridization buffer for 3 h at 68°C.

5. Hybridize with 10 ng/mL of probe (diluted in prehybridization buffer) overnight at 68°C.

6. Wash the membrane in buffer 4 for 7 min (x2) at room temperature.

7. Wash the membrane in buffer 5 for 15-20 min (x2) at 68 °C.

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