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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
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