title: Homo sapiens Hu2304 Array
status: Public on Jan 18 2007
contact.city: Delhi
contact.first: Anu
contact.last: Sharma
contact.email: anukaush@gmail.com, ramuigib@gmail.com
contact.state: Delhi
contact.address: Mall Road, Near Jubilee Hall
contact.department: Functional Genomics Unit,
contact.country: India
contact.institute: Institute of Genomics and Integrative Biology
organism: Homo sapiens
description: Number of super grid in x-direction: 2
  Number of super grid in y-direction: 1
  Total number of super grid on slide: 2
  Number of samples per grid in x-direction: 24
  Number of samples per grid in y-direction: 12
  Number of spots per grid in x-direction: 24
  Number of spots per grid in y-direction: 24
  The distance between two spots in micron: 170
  Positive control: beta-actin
  Negative control: 50% DMSO
manufacture_protocol: Around 32,000 cDNA clones were procured from M/s Genotypic technologies, Bangalore, India in the form of dried PCR products in 384 well plates. These clones were primarily obtained from Research Genetics in the form of plasmids and the inserts were amplified using degenerate primers designed against the cloning sites of 7 different plasmids used for cloning. The dried PCR products were reconstituted by re-suspending in 26 ul 1X TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0). Reconstituted PCR products were aliquoted in two 384 plates by transferring 8 ul to each replica plate using Biomek 2000 (Beckmann) robot. These products were further amplified for printing microarrays by using forward primer F1: 5'GGGTAACGCCAGGGTTTTCCCAGTCAC3' and Reverse primer R1: 5’ ACAATTTCACACAGGAAACAGCTATGACCATG 3'. The clones to be amplified for printing were robotically transferred to 96 well plate and PCR reaction was set up in these plates.
  
  50 ul PCR reaction was set up using 1 ul of reconstituted template, 0.4 pmol of each primer, 1X PCR buffer with 1.5 mM MgCl2, 200uM dNTP mix and 1.5 units Taq polymerase. The PCR conditions used were initial denaturation at 95 °C for 3min, cycling conditions 95°C for 30 secs, 62°C for 30 secs, 72°C for 3 min, for 35 cycles and a final extension step at 72 °C for 7 min. The PCR products were checked for successful amplification by gel electrophoresis on 1% agarose TAE gel.
  
  PEG 8000 (Poly ethylene glycol) was used to precipitate the amplified PCR products. Equal volumes of PEG-Sodium Acetate solution  was added to the amplified products in 96-well plate, mixed and kept in ice for at least 1 hour. It was centrifuged at 12,000 g for 15 minutes and the supernatant (containing PEG) was removed completely from each well. The pellet was washed at least twice with chilled 1ml 70% ethanol for effective removal of PEG. The pellet was air dried and dissolved in 0.1X TE. The purified products were analysed by electrophoresis on 1% agarose TAE gel.
  
  Microarrays were robotically printed using purified PCR products on aminosilane glass slides using Quackenbush protocol (Hegde et al (2000). Chip writer Pro (previously known as SDDC-2 spotter, ESI, Inc.) with SMP3 stealth pins (Telechem Inc.) was used to print all the microarray slides.
  
  The purified probes were resuspended in 50% DMSO for printing on aminosilane coated microscopic glass slides (procured from SIGMA) according to this protocol (Hegde et al, 2000). Printing of probes was carried out in a controlled environment with humidity maintained around 40%-45% and temperature of 25 °C. After completion of printing, the slides were kept inside the closed chamber of the microarray robot for at least 24 hours to allow complete drying of the spots. The spots were cross-linked to the slides by exposing slides to 200 mJ total energy of UV irradiation in a UV cross-linker (Stratagene, CA). The slides were then stored at room temperature in closed slide boxes till further use.
  
  The slides were incubated with pre-hybridization buffer (5X SSC, 0.1% SDS and 1% BSA) at 42 °C for 45 minutes for blocking the slides. The slides were washed in sterile water, rinsed in Isopropanol, air-dried and immediately used for hybridization.