title: NRC-BRI biofilms 10K v1.0
status: Public on Feb 18 2010
contact.city: Montreal
contact.first: Etienne
contact.last: Yergeau
contact.email: Etienne.Yergeau@cnrc-nrc.gc.ca
contact.state: Qc
contact.address: 6100 Royalmount Ave
contact.department: Environmental Microbiology
contact.country: Canada
contact.institute: NRC-BRI
organism: environmental samples
manufacture_protocol: Ten microliters of DNA extracted from each of the 33 samples from the Wascana Creek biofilms were pooled together to form one composite DNA sample. This composite sample was fragmented by sonication at a power of 8 W for 5 s and size-selected (400-600bp) on 2% agarose gel. Gel-extracted sonicated DNA was end-repaired using an End-it kit (Epicentre Biotechnologies, Madison, WI) and incubated with Taq DNA polymerase and ATP for 1h at 72°C to create an “A” overhang compatible with UA ligation. End-repaired DNA was then ligated in a pDrive vector (Qiagen, LaJolla, CA) and transformed into XL1-Blue competent cells (Strategene, La Jolla, CA). 9,600 clones were picked by a VersArray colony picker (Bio-Rad, Hercules, CA) at the Concordia University Centre for Structural and Functional Genomics (Montreal, QC, Canada). DNA was extracted from the clones by boiling lysis (5 min at 95°C) and was then amplified by PCR using vector-specific primers and the PCR products were purified using Multi-Screen FB 96 wells purification plates (Millipore, Billerica, MA). Following gel analysis and DNA quantification, 6543 PCR products were classified as “good” (66.2%) and 917 PCR products were classified as “weak” (9.6%). The remaining reactions (2332; 24.3%) showed either no product or multiple bands and were classified as “failed”. All PCR products were then printed in duplicate on amino-silane coated glass slides with a VersArray Chip Writer Pro printer (Bio-Rad).