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Protocol Steps  >  sample preparation step  >  Probe preparation

Probe preparation

sample preparation step

Protocol: Yeast experiment: Yeast Microarray Protocol

Indirect labeling method. Briefly, the reactive amine derivative of dUTP, 5-(3-
aminoallyl)-2?-deoxyuridine 5?-triphosphate (Sigma) is incorporated into cDNA using the Superscript II reverse transcriptase (Invitrogen) and oligo dT (Invitrogen) and random examers (Roche). After synthesis of cDNA (2–3 h at 42 °C), RNA is hydrolyzed by addition of sodium hydroxide and EDTA to a final concentration of 100 mM and 10 mM, respectively and incubated at 65 °C for 10 min. The hydrolysis reaction is neutralized with 1 M HEPES. After removing free nucleotides by purification and concentration using Microcon-30 microconcentrators, the aminoallyl-labeled samples are coupled to succinimidyl ester of cyanine-3 (Cy3) and cyanine-5 (Cy5) (Amersham) combined with 1 M NaHCO3, pH 9. Coupling takes place in the dark at 25 °C for 1 h. Appropriate Cy3 and Cy5 labeled cDNA samples are purified following Qiagen Qiaquick PCR Purification Kit instructions. Poly-dA (12–18 mer), 20× SSC, and HEPES pH 7.0 are added. After the resultant mix was filtered through a Millipore 0.45 ?M filter, 10% SDS is added. The samples are incubated for 2 min at 100 °C and cooled in a microcentrifuge prior to loading and then applied to microarray.

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