title: IMP Genomics Mouse 40MM
status: Public on Aug 08 2012
contact.city: Vienna
contact.first: Harald
contact.last: Scheuch
contact.email: scheuch@imp.ac.at
contact.state: Vienna
contact.address: Dr. Bohrgasse 7
contact.department: Core Facilities
contact.country: Austria
contact.institute: Research Institute of Molecular Pathology (IMP)
contact.phone: 00431797045512
organism: Mus musculus
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)