title: BIOTEC UniShrimp cDNA Chip [UnishrimpV8]
status: Public on Jan 02 2019
contact.city: Khlong Luang
contact.first: Wananit
contact.last: Wimuttisuk
contact.email: wananit.wim@biotec.or.th
contact.state: PATHUM THANI
contact.address: 113 Thailand Science Park
contact.country: Thailand
contact.institute: National Center for Genetic Engineering and Biotechnology
contact.phone: 6625646700
organism: Penaeus monodon
description: A cDNA microarray was constructed from the EST libraries of P. monodon, consisting of 6826 features.
manufacture_protocol: Array Design:    Amplification  Approximately 5000 sequences will be amplified either from plasmids of EST clones or from the first strand cDNA template.  For amplification from EST clones, PCR reactions were optimized considering the following parameters: (1) amounts of template, (2) types of Taq DNA polymerase, (3) units of Taq DNA polymerase, and (4) MgCl2 concentrations.   In all experiments, the reactions were performed using purified plasmids as templates in a 96-well format PCR plate (Axygen) with final concentrations of the following components: 0.3 mM dNTPs, 1X buffer, 0.4 M each primer (LM13F: CAC GAC GTT GTA AAA CGA CGG CCA G and LM13R: CAT GGT CAT AGC TGT TTC CTG T, BioBasic).  Amplification was carried out for 35 cycles at the following settings: 940C for 30 sec, 600C for 30 sec, 720C for 3 min.    For amplification from first strand cDNA, PCR reactions were optimized considering the following parameters: (1) amounts of template, (2) MgCl2 concentrations, (3) units of Taq DNA polymerase, and (4) reaction volumes.   In all experiments, the reactions were performed using cDNA as templates in a 96-well format PCR plate (Axygen) with final concentrations of the following components: 0.4 mM dNTPs, 1X buffer, 0.6 uM each gene-specific primer (designed as described below; IDT, Singapore). Amplification was carried out for 38 cycles at the following settings: 940C for 30 sec, 570C for 60 sec, 720C for 3 min.  The resulting amplicons were electrophoretically analyzed on 1.5% agarose gel prepared in 0.5X TBE buffer.  Purification  The PCR products were purified using Multiscreen PCR 96 well purification plate (Millipore, #MSNU03050). Briefly, PCR products were thoroughly loaded to the purification plate and placed under a vacuum manifold.  The bound DNA on the plate will be washed with sterile water once in vacuo until completely dried.  To collect the purified products, sterile water was added to the Millipore plate and let stand for 5-10 min before resuspended into a polysterene V-shape 96-well plate (Corning, CoStar # 3897)   Microarray fabrication    Fifteen plates of 384-well print plates were used as source plates for microarray fabrication. Source plates were resuspended in 7 uL of 3xSSC buffer (Invitrogen, cat no.15557-044) and incubated at 4C overnight before printing. The cDNA microarrays were fabricated using Nanoprint LM210 microarray spotter (ArrayIt, USA) fitted with a 24-pin head on GAPS II amino-silane coated slides (CORNING, cat no.40005). A 11,136 spots/array pattern was designed by 24 X 20 spots per block, containing a total of 24 arrays. Each amplicon was spotted in replicate. The printed slides were UV-crosslinked at 200 mJ of energy (GS Gene Linker UV chamber, BIO-RAD) and stored at room temperature in a dry incubator.