User:Karmella Haynes/Notebook/Polycomb project/2011/04/13

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04/13/11

  • ✓ qRT-PCR: Pc-TF -/+dox plates 19, 20
  • ✓ PCR: ChIP primer optimization (new primers)



qRT-PCR

> Plate 19
--> 750 nM Primers (& cDNA dilution)

  1. mCh1 (1:1000)
  2. GAPDH 21A (1:1000)
  3. ACTC1 35A (1:10)
  4. ATOH1 46D "
  5. CASP10 41A "
  6. CDKN2A 7C "
  7. EOMES 42A "
  8. GRHL2 37C "

> Templates, use 2 μL

  1. KAH154-2, 4/08/11 A
  2. KAH154-2 +dox, 4/08/11 A
  3. KAH154-2, 4/08/11 B
  4. KAH154-2 +dox, 4/08/11 B


> Plate 20
--> 750 nM Primers (& cDNA dilution)

  1. MMP12 31B (1:10)
  2. NPPA 40A "
  3. OTX2 43D "
  4. SST 45C "
  5. THPO 33A "
  6. TNF 34A "

> Templates, use 2 μL

  1. KAH154-2, 4/08/11 A
  2. KAH154-2 +dox, 4/08/11 A
  3. KAH154-2, 4/08/11 B
  4. KAH154-2 +dox, 4/08/11 B


Reagent 1 rxn Primer mixes 1-6 (x13)
diluted cDNA 2.0 ---
SYBR Green mix 7.5 97.5
750 nM primers 3.0 39.0
dH2O 2.5 32.5
  15.0

--> Aliquot 39.0 primer mix into 1st well of each 3x set
--> Add 6.0 (2.0 x3) DNA to 39.0 primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in each 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, 58°C/ 15 sec, 72°C/ 15 sec] x45
  • Melt curve range 58°C -> 95°C/ 0.5°C per step



PCR: ChIP primer optimization
> Test new ChIP primers on old DNA sample (from sonicated CH2O x-linked chromatin)

> Primers (10 μM mix)

  • 1-12. ATOH1 A1-3, B1-3, C1-3, D1-3
  • 13-24. CASP10 A1-3, B1-3, C1-3, D1-3
  • 25-36. EOMES A1-3, B1-3, C1-3, D1-3
  • 37-48. GRHL2 A1-3, B1-3, C1-3, D1-3
  • 49-60. NPPA A1-3, B1-3, C1-3, D1-3
  • 61-72. OTX2 A1-3, B1-3, C1-3, D1-3
  • 73-84. ACTC1 A1-3, B1-3, C1-3, D1-3

> Template (use 2 μL)

  • 130-4 Input, FX (#6), 9/15/10)
Reagent 1 rxn Template mix (x86)
ChIP input DNA 2.0 172.0
2x GoTaq Green mix 10.0 860.0
10 μM primers 1.0 ---
dH2O 7.0 602.0
  20.0
  • Note: use 2.0 mL tube for master mix


Bio-Rad 96-well

  • 95°C/ 3 min.
  • [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x32
  • 72°C/ 3 min.
  • 4°C/ ∞

File:KAH 041311 gel1.tif

> Conclusions: Test the following via qPCR (template vs. no template)

  • ATOH1 A1, B3, C2, D1
  • CASP10 A1, B1, C3, D2
  • EOMES A1, B2, C3, D3
  • GRHL2 A1, B1, C1, D1
  • NPPA A1, B2, C2, D1
  • OTX2 A1, B2, C1, D1
  • ACTC1 A1, B1, C2, D1