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New Packard Hybridization Protocol Uses Coverslip Air Bubbles

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Packard BioChip Technologies of Billerica, Mass., has released a new simplified protocol for microarray hybridization on glass slides. This protocol, “A short communication on microarray hybridization,” eliminates blocking steps, and encourages researchers to allow an air bubble to get under the cover slip — a practice that traditional protocols such as those recommended by the Pat Brown Lab at Stanford tell users to avoid. (See http://cmgm.stanford.edu/pbrown/protocols/). This new protocol, available on Packard’s website, www.packardbioscience.com, is designed to be easy to apply and create uniform, reproducible hybridizations. It includes the following suggestions:

• Amine or poly-(L)-lysine coated substrates are more effective than those with aldehyde attachment chemistry because the latter require additional steps in target modification. Packard uses SuperAmine glass slides from TeleChem international.

• Following deposition of cDNAs or oligos, the slides should be cross-linked using a mechanism for UV light exposure, such as Stratagene’s Stratalinker, set to 2,000 microjoules, or baked at 80 degrees Celsius for an hour.

• The blocking step using succinic anhydride should be eliminated. “Experimental data from the Packard Biochip labs routinely shows, approximately 2 out of 5 succinic anhydride treated slides have damage to data quality attributable to this step,” Packard said.

• Stationary coverslip hybridization is replaced by agitation using an air bubble under the coverslip. First, the target DNA is denatured by immersion in boiling water for two minutes, then the slide is placed in 70 percent and 95 percent ice cold ethanol for two minutes each, and air dried. Next a double adhesive slide frame seal cover slip is applied, making sure to leave an air bubble the size of a 20-point letter O. Finally, the slide is inserted into a 50 ml test tube and attached to a rotating rotor in the hybridization chamber set to 6 rpm. The agitated hybridizing mix is left to take place for 1.5 hours to overnight.

• After hybridization, the slides are washed once each in 2x salt sodium citrate (SSC), 0.1 percent sodium dodecyl sulfate (SDS) at 55 degrees Celsius, then in 2x SSC at room temperature, then in 0.2x SSC at room temperature.

— MMJ

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