title: UNIL_fireant_25K_vG_and_vI
status: Public on Oct 20 2013
contact.city: Lausanne
contact.first: Yannick
contact.last: Wurm
contact.email: yannick.wurm@unil.ch
contact.address: Biophore
contact.department: Ecology and Evolution
contact.country: Switzerland
contact.institute: University of Lausanne
organism: Solenopsis invicta
description: This platform represents two prints ("G" and "I") of the "same" microarray.
manufacture_protocol: Bacteria clones were inoculated into PCR plates containing 5 µl modified LB-ampicillin broth (20% concentration LB and without NaCl) and grown overnight. Plasmid inserts were amplified by PCR after adding 95 µl of PCR mix. A single primer, SMART PCR primer 5’-AAGCAGTGGTATCAACGCAGAGT-3’, which matches both the 3’ and 5’ SMART adapter of the inserts was used. PCR mixes contained 0.4 µl 5U/µl TAQ (Qiagen), 10 µL 10x Qiagen buffer, 20 µl Q solution, 4 µl 25 mM MgCl2, 1.5 µl 25 mM dNTPs, and 1 µl 100 µM SMART PCR primer. An initial 9-min denaturation at 94 °C was followed by 40 cycles of 30 sec at 94°C, 30 sec at 59°C, and 3 min at 72°C. The reaction ended with an additional incubation of 7 min at 72°C. Two microliters of each PCR product were analyzed on a 1% agarose gel. Gel pictures were visually examined to classify all PCR products as follows: “strong single band” (78.4 %), “no band” (3.9 %), or “weak or multiple bands” (17.5 %). This data is in Wang et al 2007  Supplemental Table F. PCR products were purified by a standard NaOAc/ethanol precipitation, resuspended in 30 µl water and transferred into duplicate 384-well plates using a Biomek FX liquid-handling robot (Beckman Coulter). Then PCR products were dried and resuspended in 20 µL 3x SSC, 1.5 M betaine. We also resuspended 48 times 10 commercial exogenous controls (SpotReport Alien cDNA Array Validation System, Stratagene) in 3x SSC, 1.5 M betaine, one set for each subgrid of the microarray. Microarrays were printed on aldehydesilane-coated slides (NexterionTM Slide AL, Schott Nexterion), using an OmniGrid 300 spotting robot (GeneMachines). Spot and printing quality were assessed visually under a dissecting microscope after printing. While a few slides had minor defects (e.g., a few spots missing or damaged by dust particles), the majority of slides exhibited no defects. DNA was crosslinked to slides by baking at 80°C for 1 h. Afterwards, the slides were post-processed with NaBH4 using the manufacturers recommended protocol.
  