title: IMP Genomics Mouse (Composite)
status: Public on Jan 21 2011
contact.city: Vienna
contact.first: Daniel
contact.last: Schramek
contact.email: daniel.schramek@imba.oeaw.ac.at
contact.address: Dr. Bohrgasse 3
contact.country: Austria
contact.institute: IMBA
organism: Mus musculus
description: Composite of three arrays (46MM, 47MM, 51MM). Design files included as supplementary data linked to GSE26763 (GSE26763_raw_gpr_design_files.tar.gz).
manufacture_protocol: Prepare in house spotted  DNA Microarrays
  
  
  PCRs and Printing
  
  Dilution plates:
  
  Prepare 96 well pates and label them ( eg. 1130 Dil)
  Aliquot 95 µl dd water into each well (roboter '95µl verteilen')
  Pipette 5µl from the copy plate into the Dilution plate (roboter 'dilution plate').
  (If you do not perform immediately:
  Heat for 10 min at 94°C ( PCR machine)
  Spin down
  Freeze away or perform PCR reaction)
  
  
  PCRmix for a 96 well plate:
  
  550µl 10x buffer (100mM Tris.HCl pH8.3, 500mM KCl)
  11µl UNI-primer (1µg/µl CCCAGTCACGACGTTG)
  11µl REV-primer (1µg/µl CACAGGAAACAGCTATG)
  11µl 1M MgCl2 (has to be adjusted to Taq used)
  44µl 25mM dNTPs
  110µl Taq
  1887µl dd water
  2514µl 3M Betaine
  5138µl total Volume (45µl per well)
  
  PCR Reaction:
  
  Transfer 5µl of the working dilution (see bacteria growth) to PCR-plate (MJResearch, skirted plate) containing 45µl PCRmix,
  
  seal plate with sealing sheet (ABGene AB-0558)
  
  Spin down
  
  PCR program:
  94°C 1min
  35X 94°C 30sec
  52°C 30sec
  72°C 1min 30sec
  72°C 5min
  
  
  Load 5µl PCR product on a 1% agarose gel for quality control
  
  
  
  Preparation for printing the PCR products:
  
  Concentrate PCR products in a Speedvac (Determine time until no further reduction of volume is seen, in our case it is at least 4 hours when using 8 plates per batch, final volume is approximately 6-7 µl)
  
  Add 6µl 6xSSC
  
  Shake vigorously up to 3 hours at 4°C (we use a modified vortex,  probably mixing with pipetting robot before transfer into 384 well plates will do)
  
  Transfer into 384 well plates
  
  Store PCR-plates at -80°C
  
  Spot onto Schott Nexterion A slides
  
  
  Slide Blocking:
  Fixation:
  Put slides 30 min on 80°C heating plate and cross link with 600mJ(Schott) or 60mJ with Poly-lysine slides
  
  Blocking:
  Dissolve 1.37 g Succinic Anhydride in 90ml N-Methyl - Pyrrolidone
  Add 2.5 ml N-methylimidazol (FLUKA)
  
  Immediately pour it into a slide chamber and incubate slides for 1 hour with slight agitation
  
  transfer slide into 0.1 % SDS for 20sec.
  
  transfer into H2O for 20sec
  
  Denature:
  Slides are briefly washed in water (or DCE) and incubated for 4 min in a boiling water bath
  
  After a brief rinse in 95% EtOH spin slides 4 min 1200rpm for drying
  
  For 30 slides in a big chamber with 300 ml blocking solution:
  (4,11 g SA in 270 ml NMP and 7,5 ml NMI)