International Center for Tropical Agriculture - CIAT, Plamira Valle del Cauca, Colombia
Manufacture protocol
Clones were derived from non-susbtractive and substractive expression libraries, employing apomictc and sexual cDNA as tester and driver samples, and vice versa. The clones were amplified by PCR. 15 μl PCR product were mixed with 15 μl spotting buffer (betaine 3M, SSC 3X) using the robot Te-Shake (Tecan Group Ltda., Männedorf, Switzerland). The mix were arrayed over poly-L-lysine covered glass slides (Erie Scientific Company) using the spotting robot SPBIO (MiraiBio Inc, Alameda, CA) at a density of 8064 spots per slide. Each PCR product were spotted once in the slide. DNA was fixed onto the slide as described (Schena, 1999). Each slide was heated at 95ºC for ten seconds and was irradiated inmediately with UV at 410mJoules. The slides were incubated at 70ºC for two hours and were stored in Darkness at room temperature until hybridization.
Description
ONLY CLONES OF INTEREST HAVE BEEN SEQUENCED AND IDENTIFIED.
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