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

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03/29/11

 * &#x2713; qRT-PCR: Pc-TF -/+ dox plates 12, 13, 14
 * &#x2713; Order oligos: RT-PCR for 4 genes from H3K27me ENCODE list; ChIP qPCR primers for new target genes
 * &#x2713; 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

--> 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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