User:Karmella Haynes/Notebook/Polycomb project/2011/04/17

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04/17/11

  • ✓ ChIP qPCR: plate ch1



ChIP qPCR
> See 12/20/11
> Set up each reaction 4x

>Plate ch1
--> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL):

  • 126-1 (16) input, pos
  • 126-1 (32) myc IP, uk
  • 126-1 (33) IgG IP, neg
  • 132-8 (21) input, pos
  • 132-8 (36) myc IP, uk
  • 132-8 (37) IgG IP, neg

--> 750 nM primers (24 rxns per primer pair):

  1. ATOH1 A1
  2. ATOH1 B3
  3. ATOH1 C2
  4. ATOH1 D1


Reagent 1 rxn Primer mix (x25)
ChIP DNA 2.0 ---
SYBR Green mix 7.5 187.5
750 nM primers 3.0 75.0
dH2O 2.5 62.5
  15.0

--> Aliquot 52.0 primer mix into 1st well of each 4x set
--> Add 8.0 (2.0 x4) DNA to 52.0 primer mix
--> Aliquot 15.0 rxn mix to other 3 wells in each 4x set

Bio-Rad CFX96 qPCR (Kirschner lab)
--> Note: Use increased annealing temp compared to previous ChIP PCR's (new primers optimized for 58°C) --> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film

  • 95°C/ 5 min.
  • [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x45
  • Melt curve range 58°C -> 95°C/ 0.5°C per step