User:Karmella Haynes/Notebook/Polycomb project/2010/10/07

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10/07/10

  • ✓ ChIP qPCR: 126-1 and 132-8 samples
  • ✓ ChIP optimization: second step (5-hour binding)


ChIP qPCR
> Optimization to make sure input gives low C(t) compared to no template ctrl.
> Set up each reaction in triplicate
> Templates (use 4.0 μL, 12 rxns each):

  1. KAH126-1 Input (#16), pos
  2. KAH126-1 αmyc IP (#18), unk
  3. KAH126-1 αIgG IP (#20), neg
  4. 0 template (dH2O)
  5. KAH132-8 Input (#16), pos
  6. KAH132-8 αmyc IP (#18), unk
  7. KAH132-8 αIgG IP (#20), neg
  8. 0 template (dH2O)

> Primers (24 rxns each):

  1. INKARF D
  2. INKARF E
  3. INKARF F
  4. GAPDH B

--> 750 nM primer mix = 30 μL 10 μM mix + 470 μL H2O


Reagent 1 rxn Primer mix (x25)
ChIP DNA (1:1) 4.5 ---
SYBR Green mix 7.5 187.5
750 nM primers 3.0 75.0
dH2O --- ---
  15.0

--> Aliquot 31.5 primer mix into 1st well of each triplicate set
--> Add 13.5 DNA to primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in 3x set

Bio-Rad CFX96 qPCR (Kirschner lab)
--> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film

  • 95°C/ 5 min.
  • [95°C/ 15 sec, 57°C/ 15 sec, 72°C/ 15 sec] x45
  • Melt curve range 57°C -> 95°C/ 0.5°C per step


ChIP optimization
> Continued from yesterday
> Make enough complete sonication buffer for bead-ab wash and washes for elution (12 mL) > Chromatin + antibody samples (1,2)
--> Add 10 μL prepped Protein A Sepharose beads
--> Rotate at 4°C/ 5 hours
> Beads + antibody samples
--> Wash off unbound antibody: pellet beads (3000 rpm/ 4°C/ 3 min.), discard sup, wash w/ complete sonication buffer (keep on ice); repeat 2x
--> Resuspend in 250 μL complete sonication buffer + 250 μL chromatin (KAH126-1 dx)
--> Rotate at 4°C/ 5 hours


> Wash and elute
--> Same protocol as αmyc-bead conjugate IP, but gently wash 3x instead of 4x
--> Elute with 60 μL 1x loading dye + 50 mM DDT (enough to load two Western wells)
--> Heat at 65°C/ 10 min., vortex every 2 min.
--> Pellet beads, transfer supernatant to new tube
--> Heat supernatant at 100°C/ 5 min.
--> Store at -20°C until Western blot.



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