title: Streptococcus agalactiae pan microarray (GBS_2603_A909_515_1)
status: Public on Nov 18 2012
contact.city: Baltimore
contact.first: Julie
contact.middle: C
contact.last: Dunning Hotopp
contact.email: jdhotopp@som.umaryland.edu
contact.state: MD
contact.address: 20 Penn St.; HSF-II; S203B
contact.department: Institute for Genomic Sciences
contact.country: USA
contact.institute: Univeristy of Maryland Baltimore
contact.phone: 410-706-5673
organism: Streptococcus agalactiae
manufacture_protocol: Group B streptococcus (GBS) amplicon microarrays were prepared using DNA fragments of the annotated open reading frames (ORF) from the 2603 strain. Additional primer pairs were designed to replace unresponsive 2603 amplicons and to include A909 strain specific ORFs. PCR primer pairs were designed with Primer3 and locally developed PERL scripts. DNA fragments were amplified with these primers using a final concentration of 1X buffer, 2.5 mM MgCl2, 0.8 mM dNTP mix (Applied Biosystems, Blanchburg, NJ, USA), 1.25 U AmpliTaq DNA Polymerase (Applied Biosystems, Blanchburg, NJ, USA), 0.15 µM each primer, and 20 ng/uL DNA with denaturation at 95 oC for 5 min followed by amplification with 35 cycles at 95 oC for 45 sec, 55 oC for 45 sec, and 72 oC for 45 sec and a final elongation step at 72 oC for 10 min. Amplicons were purified using Montage 96 well SEQ plates (Millipore, Billerica, MA, USA) and spotted onto UltraGAPS aminosaline-coated glass slides (Corning, Corning, NY, USA) using 50% dimethylsulfoxide as spotting buffer. Amplicons were bound to the slides by ultraviolet cross-linking at 25,000 µJ/cm2. Printed slides were stored until use in a bench-top desiccator.