User:Karmella Haynes/Notebook/Polycomb project/2011/03/29

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03/29/11

  • ✓ qRT-PCR: Pc-TF -/+ dox plates 12, 13, 14
  • ✓ Order oligos: RT-PCR for 4 genes from H3K27me ENCODE list; ChIP qPCR primers for new target genes
  • ✓ Cell culture: thaw 154-2, 156-5, 157-1 (for bio replicates of dox induction expt.)

qRT-PCR

> Plate 12
--> 750 nM Primers (& cDNA dilution)

  1. ATOH1 46D (1:10)
  2. TNF 34A (1:10)

> Templates, use 2 μL

  1. KAH126-1, 7/26/10 (use 1 μL, + 1 μL H2O)
  2. KAH126-1 +dox, 7/26/10 (use 1 μL, + 1 μL H2O), running low
  3. KAH128-8.3, 7/26/10
  4. KAH128-8.3 +dox, 7/26/10
  5. KAH129-4, 7/26/10
  6. KAH129-4 +dox, 7/26/10
  7. 132-8, 7/26/10
  8. 132-8 +dox, 7/26/10


> Plate 13
--> 750 nM Primers (& cDNA dilution)

  1. CASP10 41A (1:10)
  2. EOMES 42A (1:10)
  3. OTX 43D (1:10)
  4. SST 45C (1:10)

> Templates, use 2 μL

  1. KAH154-2, 7/26/10 (use 1 μL, + 1 μL H2O)
  2. KAH154-2 +dox, 7/26/10 (use 1 μL, + 1 μL H2O)
  3. KAH156-5, 7/26/10
  4. KAH156-5 +dox, 7/26/10
  5. KAH157-1, 7/26/10
  6. KAH157-1 +dox, 7/26/10
  7. FTRx, 7/26/10
  8. FTRx +dox, 7/26/10


> Plate 14
--> 750 nM Primers (& cDNA dilution)

  1. ATOH1 46D (1:10)
  2. TNF 34A (1:10)

> Templates, use 2 μL

  1. KAH154-2, 7/26/10 (use 1 μL, + 1 μL H2O)
  2. KAH154-2 +dox, 7/26/10 (use 1 μL, + 1 μL H2O)
  3. KAH156-5, 7/26/10
  4. KAH156-5 +dox, 7/26/10
  5. KAH157-1, 7/26/10
  6. KAH157-1 +dox, 7/26/10
  7. FTRx, 7/26/10
  8. FTRx +dox, 7/26/10


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



Oligos

RT-PCR
--> Are there genes associated with H3K27me3 that are not up-regulated by Pc-TF? Find out... --> Abhi extracted data from ENCODE to compare frequency of H3K27me3 (in different cell lines) within 500 bp of t.s.s.'s from Meissner's Nanostring gene set; pick five genes from top of list

  • HOXC4
  • IGF2BP2
  • NFE2
  • NEUROG1
  • SOX1


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