title: IMP Genomics Mouse 58MM
status: Public on Aug 08 2013
contact.city: Vienna
contact.first: Roman
contact.middle: R
contact.last: Stocsits
contact.email: roman.stocsits@imp.ac.at
contact.address: Campus-Vienna-Biocenter 1
contact.country: Austria
contact.institute: IMP - Research Institute of Molecular Pathology
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)??????