User:Karmella Haynes/Notebook/Polycomb project/2010/09/20

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09/20/10

  • ✓ RT-PCR: full EZH2 kd set
  • ✓ ChIP PCR: Input vs. myc IP (partial set)
  • ✓ Cell culture: expand HepG2 cells; split HEK Gal4EED into selective maintenance (2.0 μg/mL puro) and non-selective media



RT-PCR
> EZH kd set
--> Templates

  1. n.t. kd 1 8-day, 9/18/09
  2. EZH2 kd 1 8-day, 9/18/09
  3. n.t. kd 2 8-day, 9/18/09
  4. EZH2 kd 2 8-day, 9/18/09
  5. n.t. kd 4-day, 11/03/09
  6. EZH2 kd 4-day, 11/03/09
  7. n.t. kd 8-day, 11/08/09
  8. EZH2 kd 8-day, 11/08/09

--> Primers

  1. p16INK 7C (94 bp) (8 rxns)
  2. MMP12 31A (127 bp) (8 rxns)
  3. THPO 33A (103 bp) (8 rxns)
  4. TNF 34A (119 bp) (8 rxns)
  5. GAPDH 21A (100 bp), 1:1000 cDNA (8 rxns)
  6. EZH2 22A (71 bp) (8 rxns)


Reagent Single rxn
cDNA 0.5
10 μM primers 1.0
2x GoTaq Green 10.0
dH2O 8.5
  20.0 μL


--> PCR (96-well)

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
  • 72°C/ 3 min.
  • 4°C/ ∞


File:KAH 092010 gel1.tif

> Conclusions:

  • p16INK: responds slightly, but still very repressed (probably due to DNA methylation)
  • MMP12: appears to respond to EZH2 kd. Need to quantitate using Image J.
  • THPO: may respond somewhat, pretty active overall.
  • TNF: appears to respond to EZH2 kd. Need to quantitate using Image J.
  • 8-day knockdown trial #2: Need to repeat using more nt kd cDNA (loading control signal too low for good comparison)
  • EZH2: Need to repeat with less template DNA; amplification not within linear range



ChIP PCR
> Optimize template concentration
> See 6/10/10 for successful primer test
--> Templates

  1. 126-1 Input
  2. 126-1 α-myc IP
  3. 130-4 Input
  4. 130-4 α-myc IP
  5. 132-8 Input
  6. 132-8 α-myc IP
  7. 126-1 Input (1:50)
  8. 126-1 α-myc IP (1:50)
  9. 130-4 Input (1:50)
  10. 130-4 α-myc IP (1:50)
  11. 132-8 Input (1:50)
  12. 132-8 α-myc IP (1:50)

--> Primers

  1. INKARF D (135 bp) (12 rxns)
  2. GAPDH A (103 bp) (12 rxns)


Reagent Single rxn
cDNA 0.5 (1:1 cDNA)
10 μM primers 1.0
2x GoTaq Green 10.0
dH2O 8.5
  20.0 μL


--> 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 092010 gel2.tif

> Conclusions: GAPDH negative control comes down with 126-1 (double x-linked), slightly less with 132-8 (double x-linked) but not with 130-4 (single x-linked). As previously suspected, DMA plus formaldehyde may cause over-crosslinking. Continue only with single x-linked samples from now on.