title: IU/UNH/DU Daphnia pulex Generation2 3.1K
status: Public on Sep 21 2006
contact.city: Bloomington
contact.first: John
contact.middle: Kenneth
contact.last: Colbourne
contact.email: jcolbour@cgb.indiana.edu
contact.state: IN
contact.address: 915 E. 3rd St.
contact.department: Biology
contact.country: USA
contact.web_link: http://www.daphnia.cgb.indiana.edu
contact.institute: Indiana University
contact.phone: (812) 856-0099
organism: Daphnia pulex
description: The array was printed with duplicate spots side-by-side, and consisted of 24 blocks of 17 rows and 18 columns each.
manufacture_protocol: cDNA libraries were constructed as described in Colbourne et al. (submitted).  From each library, 384 samples were PCR amplified from plasmids purified using the PerfectPrep kit (Eppendorf) and sequenced using BigDye (ABI; v.2) on an ABI 3730 sequencer.  These samples served as a random sample for quality control in further analysis.  Remaining samples (3,168 in all) were PCR amplified from colony picks of transformed bacterial cells.  Template was generated by growing colonies in 1.2 ml of 2X YT and 0.005% chloramphenicol in 96-deepwell plates for 24 hours at 37 degrees C.  Reactions were run in 100 μl containing: 1X Taq buffer (Eppendorf), 0.2 mM dNTPs, 0.2 microM primers (Fwd. 5'-GTGTAAAACGACGGCCAGTAG 3’ and Rev. 5'-AAACAGCTATGACCATGTTCAC 3’), 5 U Taq (Eppendorf), and 5 microliters fresh bacterial growth or 2 microliters purified plasmid.  Quality of PCR amplifications was verified by gel electrophoresis, and number and size of bands was recorded using Kodak’s 1D imaging software (v.3.6).  Concentration of samples was determined by 96-well microplate spectrophotometer (Molecular Devices SpectraMax 190) and adjusted to 50-200 ng/microliter for printing.
  A Genemachines Omnigrid 100 was used to print the D. pulex cDNA on GapsII aminosilane slides (Corning).  The cDNA was printed in 3X SSC and 1.5 M Betaine buffer and deposited with Stealth Micro-Spotting Pins (Telechem) at 20°C and 65% humidity.  The cDNA was fixed to the microarray slides by baking at 85oC for 3 hours.  Slides were processed by agitating in 0.2 % SDS for 5 min. and in HPLC-grade water, 5 min at 21°C., followed by 2 min. in 95oC water, rinsing briefly in ice-cold 100% isopropanol, and centrifuging at 500 x g for 5 min.  Five types of negative controls were printed, including printing buffer, failed PCR reaction with template DNA but no primers, ORFs from Arabidopsis and lambda phage, and bacterial PCR spikes (Ambion).  Positive controls of D. pulex genes were printed next to printing buffer to insure no carryover, and included cytochrome c (subunits I, II, and III), cytochrome b, actin, and ferritin.