User:Karmella Haynes/Notebook/Polycomb project/2010/02/10

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02/10/10 ✓ RT-PCR: Polycomb-ATF lines vs. EZH2 k.d. ✓ HEK293 #4 transfection: refresh medium RT-PCR --> Primers: (Based on RT-PCR trials from 11/5 and 11/9) MYT1 (1D) p15INK (5) RUNX3 (8A) TGFB1 (10A) PLAU (12A) KRT17 (14A) LAMB3 (16A) GAPDH loading control (21A) --> cDNA Template dilutions KAH126-1 (hPc-CD): a. 1:1, b. 1:100, c. 1:10,000 KAH130-2 (hCBX8): a. 1:1, b. 1:100, c. 1:10,000 EZH2 k.d. (9/18/09): a. 1:1, b. 1:100, c. 1:10,000 n.t. siRMA (9/18/09): a. 1:1, b. 1:100, c. 1:10,000 Row cDNA sample Col. 1-3 4-6 7-9 10-12 A KAH126-1 (hPc-CD) (1D) + a, b, c (5) + a, b, c (8A) + a, b, c (10A) + a, b, c B KAH126-1 (hPc-CD) (12A) + a, b, c (14A) + a, b, c (16A) + a, b, c (21A) + a, b, c C KAH130-2 (hCBX8) (1D) + a, b, c (5) + a, b, c (8A) + a, b, c (10A) + a, b, c D KAH130-2 (hCBX8) (12A) + a, b, c (14A) + a, b, c (16A) + a, b, c (21A) + a, b, c E EZH2 k.d. (11/05) (1D) + a, b, c (5) + a, b, c (8A) + a, b, c (10A) + a, b, c F EZH2 k.d. (11/05) (12A) + a, b, c (14A) + a, b, c (16A) + a, b, c (21A) + a, b, c G n.t. siRNA (11/05) (1D) + a, b, c (5) + a, b, c (8A) + a, b, c (10A) + a, b, c H n.t. siRNA (11/05) (12A) + a, b, c (14A) + a, b, c (16A) + a, b, c (21A) + a, b, c Reagent Volume DNA mix (x9) Primer mix (x13) cDNA 0.5 4.5 (9 x 0.5) --- 10μM primers 1.0 --- 13.0 (13 x 1.0) 2x GoTaq Green 10.0 --- --- dH2O 8.5 40.5 (9 x 4.5) 52.0 (13 x 4)   20.0 μL --> Aliquot 5.0 μL of each DNA mix to appro. wells --> Aliquot 5.0 μL of each primer mix to appro. wells --> Add 10.0 GoTaq to each well --> PCR (96-well) 95°C/ 3 min. [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35 72°C/ 3 min. 4°C/ ∞ --> Looks okay. Use 1/2 as much EZH2 k.d. cDNA next time. --> Repeat this expt. with cells induced w/ dox for 8 days (vs. no dox).