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

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

1. K562 Sample 1: 0.44 (260), 2.043 (260/280), 352.1 (ng/mL)
2. K562 Sample 2: 0.385, 2.035, 308.3
3. SK-N-SH Sample 1: 0.054, 1.819, 43.3
4. SK-N-SH Sample 2: 0.201, 2.016, 160.7

• Need to make 8μL solutions of the lowest concentration for each cell line

1. Sample 1: 7.1μL cells + 0.9 H2O = 2.5ug
2. Sample 2: 8μL cells = 2.5ng
3. Sample 3: 8μL cells = 0.35ug
4. 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:

1. Stage 1: 50 min @ 50°C
2. Stage 2: 5 min @ 85°C
3. Stage 3: Infinity @ 4°C

• Add 1μL: of RNase H
• Incubate @ 37°C for 20 min
• Store at -20°C