Haynes Lab:Notebook/Synthetic Biology and Bioinformatics for Predictable Control of Therapeutic Gene2/2013/01/10

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cDNA Preparation Work with samples on ice Ice: RNase H, SS III RT, and RNase out Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT Sample Reading Add 2μL of each sample plus blank to the plate reader K562 Sample 1: 0.44 (260), 2.043 (260/280), 352.1 (ng/mL) K562 Sample 2: 0.385, 2.035, 308.3 SK-N-SH Sample 1: 0.054, 1.819, 43.3 SK-N-SH Sample 2: 0.201, 2.016, 160.7 Need to make 8μL solutions of the lowest concentration for each cell line Sample 1: 7.1μL cells + 0.9 H2O = 2.5ug Sample 2: 8μL cells = 2.5ng Sample 3: 8μL cells = 0.35ug Sample 4: 2.2μL cells + H2O = 0.35ng Part 1 Make sure all samples are filled up to 8uL (fill rest with water if not) Add 1μL of primer (Oligo dT) Add 1μL of dNTP Incubate for 5 min @ 65°C Ice for 1 min Part 2 Make a MM for 4 rxns: need 2μL RT Buffer, 4μL MgCl2, 2μL DTT, 1μL RNaseOut, and 1μL SS III per rxn Add 10μL of MM to each rxn tube Mix tubes gently, centrifuge for a few seconds Part 3 RT-PCR Machine, set up as follows: Stage 1: 50 min @ 50°C Stage 2: 5 min @ 85°C Stage 3: Infinity @ 4°C Add 1μL: of RNase H Incubate @ 37°C for 20 min Store at -20°C