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)

  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


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/ ∞

File:KAH 021010 gel1.tif

--> 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).