User:Karmella Haynes/Notebook/Polycomb project/2010/09/29

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09/29/10

  • ✓ HEK Gal4EED cells: died (neglected cells for too long, restart culture); request vial from Mirra
  • ✓ ChIP-PCR: all α-myc and α-IgG IP's



ChIP PCR
> Note: dx = DMA/CH2O double x-linked; fx = CH2O x-linked
--> Templates (use 1 μL and 2 μL 1:50 dilutions of each template; 20 rxn's total)

  1. (8) 130-4 α-myc IP
  2. (10) 130-4 α-IgG IP
  3. (3) 126-1 dx α-myc IP
  4. (5) 126-1 dx α-IgG IP
  5. (18) 126-1 fx α-myc IP
  6. (20) 126-1 fx α-IgG IP
  7. (13) 132-8 dx α-myc IP
  8. (15) 132-8 dx α-IgG IP
  9. (23) 132-8 fx α-myc IP
  10. (25) 132-8 fx α-IgG IP

--> Primers

  1. INKARF D (135 bp) (20 rxns)
  2. GAPDH B (103 bp) (20 rxns)


Reagent 1 μL template 2 μL template
ChIP DNA 1.0 2.0
10 μM primers 1.0 1.0
2x GoTaq Green 10.0 10.0
dH2O 8.0 7.0
  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/ ∞


ChIP PCR 9/29/10

> Conclusions:

  • Double x-linked samples are very non-specific; do not use
  • Single x-linked samples show mostly no pull-down for GAPDH neg ctrl.
  • 126-1 appears to IP with INKARF D
  • Next ChIP-PCR: compare antibody IP vs. supernatant to get ratios of enrichment before running qPCR



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