title: BIOTEC Penaeus monodon_ReproArray_Printrun2_3
status: Public on Oct 01 2009
contact.city: Pathumthani
contact.first: nitsara
contact.last: karoonuthaisiri
contact.email: nitsara.kar@biotec.or.th
contact.state: Pathumthani
contact.address: Paholyothin
contact.department: Central
contact.country: Thailand
contact.institute: BIOTEC
contact.phone: 6625646700
organism: Penaeus monodon
manufacture_protocol: EST Library construction
  The P. monodon testes and ovary libraries were previously constructed using a ZAP-cDNA synthesis and cloning kit (Strategene, #200400, La Jolla, CA).  Briefly, cDNAs with fragments length at least 500 bp were cloned into dephosphorylated EcoRI/XhoI-digested Uni-ZAP XR and transfected into E.coli XL1-Blue MRF’.  The resulting lambda library was converted into a pBluescript library by in vivo excision according to the manufacturer’s protocol.  The ovary library was further normalized to remove abundant transcripts.  In addition, subtractive ovary libraries between mature and immature ovaries were successfully constructed (Leelatanawit et al., 2004).
  Amplication of cDNAs and microarray construction
  Amplification
  cDNA inserts were amplified via polymerase chain reactions (PCRs) in 96-well plates using GeneAmp PCR system 9700 (ABI, Foster City, CA).  For the testes library, templates were replicated from cultures grown in 96-well plates containing 160 l liquid 2x YT with 7.5% glycerol and 100 g/ml Ampicilin at 370C, covered with AirPore membranes (Qiagen, #19571, Valencia, CA) while shaking  for 16-18 hrs.  For the ovary library, plasmid stocks (approx. 10 ng each) were used as templates.  A PCR reagent master mix consisting of 0.2mM dNTPs, 1x PCR buffer, 1.5 mM MgCl2, 0.5 mM each primer (LM13F: CAC GAC GTT GTA AAA CGA CGG CCA G and LM13R: CAT GGT CAT AGC TGT TTC CTG T), and 4.5 U Taq polymerase was aliquoted to each well.  The PCR reactions were carried out using the following conditions: denaturation at 940C for 3 min, 35 cycles at the following settings: 940C for 25 sec, 600C for 25 sec, 720C for 3 min, and a final extension of 720C for 7 min.  The resulting amplicons were electrophoretically analyzed on 1% agarose gel prepared in 1x TBE buffer.
  Purification
  Purification of the amplicons was performed using GF/B filter 96-well purification plates (Whatman, #7700-1303, Florham Park, NJ).  Briefly, equal volumes of binding buffer (7M Guanidine-HCL; 200mM MES Buffer, pH 5.6) and PCR product were thoroughly mixed before transferred to the purification plate and placed under a vacuum manifold.  The bound DNA on the plate was washed with 200 l of 80% ethanol twice under vacuum until dried.  Ethanol trace was further removed by centrifuging at 1,500 rpm for 10 min.  To elute the purified products, a new standard 96-well plate was placed underneath before adding 200 l of water to the purification plate and let stand for 5-10 min before centrifuged at 1,500 rpm for 10 min.  The concentrations and yields of the purified products were assessed by agarose gel electrophoresis and quantified using a spectrophotometer.  A typical yield after purification was 2-4 g per reaction.
  The purified PCR products in 96-well plates were air-dried until complete absence of liquid.  The plates were resuspended with 8 l 3xSSC, covered tightly with foil and left in 40C overnight.  The resuspended products were transferred from 96-well plates to 384-well plates, covered tightly with foil left in 40C a day prior to microarray fabrication.
  Microarray fabrication
  The 4,992 PCR products (1920 and 3072 products from the ovary and testes libraries, respectively) were spotted onto aminosilane UltraGAPS slides (Corning, #40017, Big Flats, NY) using an automatic spotter, SpotArray 72 (PerkinElmer, Waltham, MA).  All arrays contained 16 blocks (4 x 4 configurations) of spots.  Each block had 15 rows and 21 columns.  The fabricated slides were stored in a humidity-controlled cabinet until used.
  DNA Sequencing and Analysis of testes library
  For the testes library, the purified amplicons from the colony PCR were sequenced unidirectionally using an automated ABI 3730 DNA sequencer (GE Healthcare, Foster City, CA).  Nucleotide sequences were analyzed using ESTplus, an integrative system for comprehensive and customized EST Analysis and proteomic data matching (Pacharawongsakda et al., 2008).  Briefly, the sequences were compared by BLASTX with the GenBank nr database.  Significant probabilities of matched nucleotides were considered when E-value was < 10-5.  Blast2GO was used to analyze gene ontology further.
  Microarray Post-Processing
  Prior to hybridization, the arrays were post-processed according to the recommended protocol by Corning for UltraGAPS slides.  Briefly, rehydration by passing the slide over water steam for 3 sec and snap drying on a 700C heat block allowed denaturing spotted double-stranded probes to evenly spread within their spotted areas.  The arrays were immobilized by a UV cross-linker before blocking the unused surfaces.  The arrays were immersed in blocking solution (5x SSC, 0.1% SDS, and 0.1 mg/mL BSA) at 420C for 45-60 minutes before washing with 0.1x SSC at room temperature for two times and final rinsing with MiliQ-water for 30 sec.  The arrays were dried by centrifugation at 1,600 rpm for 2 min.
  Test Hybridizations
  Quality and spot morphology of arrays from different locations from the same and different print-runs were tested using a DNA probe amplified from pBluescript using LM13F and LM13R primers.  The probe was labeled with AlexaFluor® 546 using ARES DNA labeling kit (Invitrogen, #A21667, Carlsbad, CA).  The probe was resuspended in hybridization solution (50% Formamide; 5x SSC; 0.1% SDS; 1 mg/ml Human Placental DNA; 10mM Tris, pH 7.5) and placed onto the post-processed slides.  The slides were hybridized at 420C for 16 hrs before they were washed according to Corning’s protocol.