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

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04/13/11

 * &#x2713; qRT-PCR: Pc-TF -/+dox plates 19, 20
 * &#x2713; 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

--> 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)


 * 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/ ∞



> 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


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