User:Karmella Haynes/Notebook/Polycomb project/2010/09/19

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09/19/10

  • ✓ RT-PCR: EZH2 kd cDNA, NS candidate genes; re-do MMP12, THPO, TNF in NS cDNA set (1:10 dilutions)



RT-PCR
> EZH kd set
--> Templates

  1. n.t. siRNA 4-day, 11/03/09
  2. EZH2 kd 4-day, 11/03/09
  3. n.t. siRNA 8-day, 11/03/09 (?)
  4. EZH2 kd 8-day, 11/03/09 (?)

--> Primers

  1. p16INK 7C (94 bp) (4 rxns)
  2. MMP12 31A (127 bp) (4 rxns)
  3. THPO 33A (103 bp) (4 rxns)
  4. TNF 34A (119 bp) (4 rxns)

> NS cDNA set
--> Templates (1:10 dilutions)
--> Primers

  1. MMP12 31A (127 bp) (16 rxns)
  2. THPO 33A (103 bp) (16 rxns)
  3. TNF 34A (119 bp) (16 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/ ∞

RT-PCR 9/19/10

> Conclusions:

  • EZH2 kd: looks like genes are de-repressed after EZH2 kd, but p16INK is very weak. Do PCR again on full set of EZH2 kd's
  • Pc-ATF repeats: choose best trial for figure (vs. previous trial with more template)



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