User:Karmella Haynes/Notebook/Polycomb project/2010/06/10

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06/10/10

  • ✓ ChIP: 126-1 and U2OS plain samples (corrected protocol)
  • ✓ PCR: test ChIP primers on U2OS plain quality control DNA



ChIP
> Do 4 IP's for each sample
> Samples:

  1. 126-1, ~1.7 mL/ IP
  2. U2OS plain (remainder from last attempt), ~1.2 mL/ IP

> Antibodies for IP

  1. mouse α-PolII 8WG16 (5 μg)
  2. rabbit α-myc ab9106 (5 μg)
  3. rabbit α-H3K27me3 07-449 (5 μg)
  4. mouse α-IgG sc-2025 (2 μg) + rabbit α-IgG sc-2027 (2 μg)



PCR
> Test ChIP PCR primers (expected amplicons in bp)

  1. INKARF A f1/r1 = 125
  2. INKARF B f1/r1 = 134
  3. INKARF C f1/r1 = 138
  4. INKARF D f1/r1 = 135
  5. INKARF E f1/r1 = 86
  6. INKARF F f1/r1 = 100
  7. INKARF G f1/r1 = 115
  8. INKARF H f1/r1 = 113
  9. INKARF I f1/r1 = 110
  10. ATF3 A f1/r1 = ?
  11. CCND2 A f1/r1 = ?
  12. GAPDH A f1/r1 = 117
  13. GAPDH B f1/r1 = 128

> Templates

  1. U2OS sonicated DNA quality control sample (from 5/11/2010)
  2. Flp-in T-REx genomic DNA


Reagent Volume Master mix (x14) File:KAH 061010 gel1.tif
15 μL/lane; 2% agarose
DNA 0.5 7.0
10μM primers 1.0 ---
2x GoTaq Green 10.0 140.0
dH2O 8.5 119.0
  20.0 μL
 
 
 
 

--> Aliquot 19.0 μL of each master mix to appro. wells
--> Add 1.0 primers 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/ ∞

--> Success! All primer pairs work. DNA from sonicated chromatin can be amplified by all primer pairs.