title: Sanger Institute M. musculus array, Mver1.1.1
status: Public on Mar 12 2006
contact.city: Brussels
contact.first: Jean-Baptiste
contact.last: Demoulin
contact.address: av Hippocrate 75, B1.74.05
contact.department: de Duve Institute
contact.country: Belgium
contact.institute: Université catholique de Louvain
organism: Mus musculus
manufacture_protocol: 1. Inoculate media (200 ul/well of L-broth supplemented with 7.5% glycerol and 50 ug/ml ampicillin) in 96-well microtitre plates (Falcon, 353072) with bacterial clones. Incubate for 16 hours at 37C. These cultures will represent the working copy of the clones from which all subsequent manipulations are performed. 2. Perform a 1:10 dilution of the bacterial cultures by removing 20 ul from each well and add into 180 ul of sterile ddH20 in a new microtitre plate. Store the remaining working copy plates at -70C. The dilutions can now be used immediately or stored at -20C. 3. For each plate of clones to be PCR amplified, set up PCR reactions in 96-well thin-walled thermowell plates (Costar, 6511) by mixing the following on ice: 15 ul of 1:10 bacterial culture dilution, 6 ul of 10x PCR buffer (500 mM KCl, 50 mM Tris pH 8.5, 25 mM MgCl2), 3 ul of dNTPs (10 mM dATP, dTTP, dCTP, dGTP), 1.5 ul of 5'-aminolink primer 1 (sense, 200 ng/ul), 1.5 ul of primer 2 (antisense, 200 ng/ul), 0.375 ul of Taq polymerase (5 U/ul, Perkin Elmer-Cetus), and 32.625 ul of sterile ddH20 (total volume = 60.0 ul). 4. Place PCR reactions in thermocycler and incubate for 10 min at 95C, and follow with the program - 95C for 1 min; 60C [1] for 1.5 min; 72C for 7 min - for 30 cycles. Finish with a 10 min incubation at 72C. 5. Analyse 2 ul of PCR products [2] on 2.5% agarose 1x TBE slab gels. 6. Transfer PCR products to Multiscreen filter plates (Millipore, MAGVN2250) and add 15 ul of 4x spotting buffer (1 M sodium phosphate buffer pH 8.5, 0.001% sarkosyl) to each PCR reaction. Filter by centrifugation at 2000 rpm for 10 min into 96-well plates (Falcon, 353077). Samples are now ready to array or can be stored at -20C. Notes: [1] Annealing temp may vary depending on the primer pairs used. [2] The PCR products should be at a final concentration of between 100-200 ng/ul.