title: DKFZ_LION Mouse 20k cDNA array
status: Public on Jun 10 2006
contact.city: Heidelberg
contact.first: Hendrik
contact.last: Reuter
contact.email: Hendrik.Reuter@beiersdorf.com
contact.address: INF280
contact.department: Moleculare Genetics B060
contact.country: Germany
contact.institute: DKFZ
contact.phone: +496221424677
organism: Mus musculus
manufacture_protocol: Spotted cDNA array produced using the commercially available 20K library arrayTAG; LION bioscience AG, Heidelberg, Germany. Inserts were amplified using the M13 primers 5 -TCG AGC GGC CGC CCG GGC AGG T-3  and 5 -Amino(C6)-AGC GTG GTC GCG GCC GAG GT-3  (Biospring, Frankfurt, Germany). For each cDNA clone, 1  l bacterial suspension of an overnight culture was used as template in a 40 ul PCR reaction containing 10 mM Tris-HCl (pH 8.3 at 20°C), 50 mM KCl, 1.5 mM MgCl2, 0.4  M of each primer, 0.2 mM of each dNTP and 1.2 U AmpliTaq DNA polymerase (Applied Biosystems). The amplification reaction was performed in a Primus HT Multiblock PCR-System (MWG Biotech, Ebersberg, Germany): an initial denaturation step at 94°C for 2 min was followed by 33 cycles of denaturation at 94°C for 15 sec, annealing at 68°C for 30 sec and extension at 72°C for 50 sec, with a final extension step at 72°C for 5 min. Purity and quantity of the PCR products were analysed by electrophoresis on 1.2% 96-well-Ready-to-Run-Agarose-Gels (Amersham Biosciences) according to the manufacturer's instructions. For a subset of the clones, DNA quality and concentration were evaluated by absorption spectroscopy between 220 and 320 nm (1 OD260 = 50  g/ml dsDNA). PCR products were purified by ethanol precipitation (2.5 vol. 100% ethanol; 1/10 vol 3 M sodium acetate, pH 5.2), followed by a washing step with 70% (v/v) ethanol. Pellets were dried, dissolved in 20  l of 3xSSC, 1.5M betaine and transferred to 384-well-microtiter plates applying the robotic liquid handling system MultiProbe II EX (Packard Bioscience, Dreieich, Germany). Dissolved PCR products were spotted as array duplicates using epoxy-silane coated glass slides (Schott Nexterion) and stainless steel split pins (SMP3, Telechem) at 20°C and 40% rH. Slides were dried (60' @ 60°C) and DNA was UV-crosslinked with 240 mJ/cm2 @ 254 nm. Slides were stored, sealed airtight and dry, at 4°C.