title: DeRisi_8Mer_1457_v1
status: Public on Aug 29 2010
contact.city: San Francisco
contact.first: Polly
contact.last: Fordyce
contact.email: polly@derisilab.ucsf.edu
contact.state: CA
contact.address: 1700 4th St, Byers Hall Room 403
contact.country: USA
contact.institute: UCSF
organism: synthetic construct
description: Cy5-labeled dsDNA was diluted to a final concentration of 1.25 μM in 3× SSC with 0.125% polyethylene glycol (PEG) (Fluka) and 12.5 mg/mL d-(+)-trehalose dihydrate (Fluka) (for enhanced subsequent solubility) and printed onto custom 2″ × 3″ ThermoFisher Scientific SuperChip Epoxysilane slides (ThermoFisher Scientific) using a DeRisi lab custom microarrayer with 75 um silicon tips.  Arrays were printed using 12 tips in a single row with a 65 spot sector width, 320 um column spacing, and 900 um row spacing.
manufacture_protocol: Sequences were hybridized to a single Cy5-labeled oligonucleotide (5'-Cy5-GTCATACCGCCGGA-3') (IDT) and extended using Klenow fragment (exo-) (New England Biolabs) to produce Cy5-labeled dsDNA. Final reaction conditions were 100 uM each dNTP, 5 uM Cy5-labeled oligonucleotide, 5 uM library oligonucleotide, and 2 units of Klenow fragment (exo-) in 1x NE Buffer 2.  Reactions were initally prepared without Klenow fragment (exo-), denatured at 94C for 3 min, and cooled to 37C over 45 min.  Following addition of Klenow fragment (exo-), reactions were incubated at 37C for 1 hour to allow complete extension, following by heat inactivation of the enzyme at 72C for 20 minutes and slow cooling (45 min) back to 10C.