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)??????
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