User:Karmella Haynes/Notebook/Polycomb project/2011/04/07

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04/07/11

 * &#x2713; qRT-PCR: Pc-TF plate 17
 * &#x2713; RNA preps: 129-4, 154-2, 156-5, 157-1 -/+dox
 * &#x2713; Repr. luc reactivation expt.: wash out dox from 2 and 4-day plates; split all 1:2

qRT-PCR

> Plate 17 --> 750 nM Primers (& cDNA dilution)
 * 1) mCh1 (1:1000)
 * 2) GAPDH 21A (1:1000)
 * 3) ACTC1 35A (1:10)
 * 4) GRHL2 37C "
 * 5) NPPA 40A "
 * 6) CDKN2A 7C "
 * 7) MMP12 31B "
 * 8) THPO 33A "

> Templates, use 2 μL
 * 1) KAH132-8, 6/18/10
 * 2) KAH132-8 +dox, 6/18/10
 * 3) KAH132-8, 6/23/10
 * 4) KAH132-8 +dox, 6/23/10

--> 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

RNA Preps > Resuspend in 15 μL THE RNA storage solution; store at -80°C until cDNA synthesis


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