User:Karmella Haynes/Notebook/Polycomb project/2010/10/02

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10/02/10

  • ✓ ChIP qPCR optimization: Optimization PCR



qPCR
> Optimization
> Set up each reaction in triplicate > Templates (use 1.0, 2.0, 4.0, and 0 μL):

  • KAH126-1 Input, positive (1:50)
  • KAH126-1 αIgG IP, negative (1:50)

--> Make master mix of 13x for each template at each volume; 8 "mixes" total
> Primers:

  • INKARF D (24 rxns)
  • INKARF E (24 rxns)
  • INKARF F (24 rxns)
  • GAPDH B (24 rxns)

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


Reagent 1 rxn Primer mix (x25)
ChIP DNA (1:50) up to 4.5 ---
SYBR Green mix 7.5 187.5
750 nM primers 3.0 75.0
dH2O --- ---
  15.0

Bio-Rad CFX96 qPCR (Kirschner lab)
--> Use Bio-Rad 96-well low profile plate MLL-9601 + clear 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/ 3°C per step


> Conclusions:

  • ~1 C(t) increase per doubling of template amount. Average C(t) for 4 μL template reactions (Input pos ctrl) is about 26 - 36 (a little too weak, neg ctrl is ~40). Use 4.5 μL of 1:25 dilution for future reactions.
  • Separate pipetting of template into primer mix yeilds too much variation btwn tech replicates. Mix 3 rxns in one tube (45 μL), then aliquot 15 μL.