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

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

02/10/10

 * &#x2713; RT-PCR: Polycomb-ATF lines vs. EZH2 k.d.
 * &#x2713; HEK293 #4 transfection: refresh medium

RT-PCR --> Primers: (Based on RT-PCR trials from 11/5 and 11/9)
 * 1) MYT1 (1D)
 * 2) p15INK (5)
 * 3) RUNX3 (8A)
 * 4) TGFB1 (10A)
 * 5) PLAU (12A)
 * 6) KRT17 (14A)
 * 7) LAMB3 (16A)
 * 8) GAPDH loading control (21A)

--> cDNA Template dilutions
 * 1) KAH126-1 (hPc-CD): a. 1:1, b. 1:100, c. 1:10,000
 * 2) KAH130-2 (hCBX8): a. 1:1, b. 1:100, c. 1:10,000
 * 3) EZH2 k.d. (9/18/09): a. 1:1, b. 1:100, c. 1:10,000
 * 4) n.t. siRMA (9/18/09): a. 1:1, b. 1:100, c. 1:10,000

--> 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).


 * }