title: Zebra mussel byssus cDNA microarray
status: Public on Apr 19 2008
contact.city: Chicago
contact.first: Wei
contact.last: Xu
contact.email: wei-xu@northwestern.edu
contact.state: IL
contact.address: 303 E Chicago Ave, Tarry 4-714
contact.department: Surgery
contact.country: USA
contact.institute: Northwestern University, Feinberg School of Medicine
contact.phone: 312 908 0566
organism: Dreissena polymorpha
manufacture_protocol: 716 E.coli clones were selected from thirty-four 96-well plates and re-arranged into eight new 96-well plates. The overnight cultures from the eight 96-well plates were used to prepare the PCR templates. 10 μl of each bacterial culture was taken to mix with 90 μl sterile double distilled water. Then the mixture was incubated at 100 ℃ for 10 minutes followed by 15 minutes 1200g centrifuge. The supernatant was used as PCR templates. M13 forward (5’- GTT TTC CCA GTC ACG ACG TTG -3’) and reverse (5’- TGA GCG GAT AAC AAT TTC ACA CAG -3’) primers were applied with GoTaq Green Mastermix (Promega, Madison, WI) to yield 100 μl PCR product for each EST. PCR products purification was done by MinElute 96 UF PCR Purification Kit (Qiagen, Valencia, CA). All the purified PCR products were dissolved with 20 μl 50% dimethyl sulfoxide (DMSO). 1 μl of each purified amplicon was loaded on 1.2% agarose TAE gel to detect the presence of the products. All the purified amplicons were transferred to two 384 well plates by Tecan Genosys 150 liguid handling robot (Tecan US, Research Triangle Park, NC) before the microarray spotting.
  The cDNA microarray spotting was performed by GeneTAC G3 arraying robot (Genomic Solutions, Ann Arbor, MI) which is equipped with a 48-pin head. The cDNA microarray designed for the 716 genes contains 48 sub-arrays (4 ×12). In each sub array, 64 spots were distributed as an 8 × 8 matrix. The 716 amplicons were spotted in triplicates, which are randomly located in each sub-array. Zebra mussel β-actin was used as positive control spotted in every subarray while the spotting solutions were used as negative controls in each subarray. Meanwhile, twelve spots in each sub-array are left blank for background control and negative control as well.