title: Lactobacillus johnsonii NC533 microarray project
status: Public on Jan 17 2008
contact.city: Lausanne
contact.first: Emmanuel
contact.last: Denou
contact.email: emmanuel.denou@rdls.nestle.com
contact.state: vaud
contact.address: vers chez les blancs
contact.department: Nutrition & Health
contact.country: Switzerland
contact.institute: Nestlé Research Center
contact.phone: + 41 21 785 9315
organism: Lactobacillus johnsonii NCC 533
manufacture_protocol: Primer synthesis of 1876 pairs of primers in 96 well Multiplex format
  Each Forward primer carries a Tag sequenceand a total number of 9 different tags have been designed for covering all Forward primers. All reverse primers contains a unique tag
  Following a 96 well plate map, a specific arrangement of these 9 tags provides a way to eliminate one major source of DNA contamination during the secondary amplification by locating one tagged PCR product away from the adjacent one that will be amplified with identical tags. Basically, tags 1 to 9 are arranged in a row starting from position A1 to A9, and this is repeated in the next positions and second lane of the plate, i.e. well plates B1 and B2 will contain primers tagged with 4 and 5, respectively
  Primary PCR amplification: The reverse primers were mixed with the corresponding Forward primers and diluted each to 5 mM final concentration to prepare a 10-fold concentrated solution of specific primers
  A master mix is prepared with EuroTAQ incubation buffer, EuroTAQ thermostable polymerase (EGT), 0.2 mM each dNTPs (EGT), 1.8 mM MgCL2, LA-1 genomic DNA (5 ng), and dispensed by 90 µl to the 96 well PCR plates.
  PCR amplification were performed in Applied Biosystem thermocycler GeneAmp PCR system 9700 with 1 min at 94°C for initial denaturation followed by 30 cycles (94°C, 1 min; 50°C, 30 sec; 72°C, 1 min), and finally a final step at 72°C for 5 min.
  An aliquot of each PCR mix (5 µl) was resuspended in EthBr loading buffer and analyzed by agarose gel electrophoresis (1% in TAE buffer).
  The size of amplicons were determined with the help of  the molecular weight DNA ladder Smart Ladder (EGT).
  Secondary PCR amplification : The unique Reverse Tag primer was mixed with the corresponding Forward primers 1 to 9 in order to prepare a template 96 well plate that was used for transferring the tag primer couples at the good locations in all 96 well PCR plates (10 µl, 5 mM final concentration)
  We obtained 98.6% of the aminated secondary PCR products, which covers 1876 ORFs
  PCR purification was performed by using the Millipore Multiscreen 96 well Filter Plates.
  Based on differential binding of DNA molecules on silica matrix, this system is adapted for centrifugation with a throughput of 17 plate per centrifuge.
  A binding buffer (Guanidine-HCl7 M; MES 200 mM pH = 5.6) is first added to the PCR plates.
  Following up and down pipetting, the samples are transferred to the purification plate for binding onto the glass fibers during centrifugation.
  One step centrifugation washing is done with Ethanol 80 %. Amplicons are recovered in H20 buffered at pH 8 (120 µl).
  Water have been removed by evaporation and DNA samples were resuspended to a final volume of 30 µl 3x SSC printing buffer (average concentration 100-200 ng/µl).
  An aliquot of each first lane (A1 to A12 well) from 96 well plates (1 µl) was resuspended in EthBr loading buffer and analysed by agarose gel electrophoresis (1% in TAE buffer).
  For arraying the aminated PCR products, the samples were transferred from 96 well plates to V-bottom 384 well plates (Genetix #7020).
  Additional controls (printing buffer, C1, C2, C3 and luciferase cDNA from Renilla) were added in the last 384 well plate in order to place these controls in each subgrid of the array (16 subgrids in total).
  We used the Virtek ChipWriterPro, equipped with 16 SMP3 quill pins (Array It, Telechem) for arraying the library in close duplicates with 200 µm dot spacing onto CMT-GAPS II Corning glass slides.
  Each subgrid contains 16 columns and 16 rows.
  In order to provide an area for normalising the laser intensities for working at an optimal dynamic range and the scans for normalisation on intensities recovered from spike control RNA (LuxA control RNA available at Promega), we added an extra lane located outside of the main array frame that contains serial two-fold dilutions of sonicated LA-1 genomic DNA, Luciferase cDNA and CAT cDNA.
  Following printing, the slides were dried overnight at room temperature in the gridder chamber and processed according to the Anhydride succinic acid protocol recommended by Corning.
  Printed microarray slides were incubated in MilliQ water for 2 min at 95°C for DNA denaturation.
  For checking the quantity and quality of DNA spotted onto the glass slides, we performed a Cy5-dCTP Terminal Transferase labelling (Roche) of bound DNA molecules onto the ready-to-use LA-1 microarray glass slides (one slide per 40 slides-batch).
  For this purpose, we directly added to the printed glass slide area, 20 µl of dCTP labelling reactional mix according to the manufacturer’s recommendations.
  A coverslip was applied onto the array and the glass slide was incubated for 15 min at 37°C.
  The glass slide was then washed 5 min in 0.2x SSC, 0.1% SDS and 5 min in 0.2x SSC and finally spin-dried.
  Microarray glass slides were scanned with Genepix 4000 scanner (Axon instruments, Inc.) and arrays analysed with Genepix 4.0 software.