User:Karmella Haynes/Notebook/Polycomb project/2011/03/24

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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03/24/11

 * &#x2713; qRT-PCR: Pc-TF -/+ dox plate 7, 8

qRT-PCR

> Plate 7 --> 750 nM Primers (& cDNA dilution)
 * 1) mCh1 (1:1000)
 * 2) GAPDH 21A (1:1000)
 * 3) ACTC1 35A (1:10)
 * 4) GRLH2 37C (1:10)

--> Templates, use 2 μL
 * 1) KAH126-1, 6/15/10
 * 2) KAH126-1 +dox, 6/15/10
 * 3) KAH132-8, 6/15/10
 * 4) KAH132-8 +dox, 6/15/10
 * 5) KAH154-2, 6/15/10
 * 6) KAH154-2 +dox, 6/15/10
 * 7) FTRx, 6/15/10
 * 8) FTRx +dox, 6/15/10

> Plate 6 --> 750 nM Primers (& cDNA dilution)
 * 1) NPPA 40A (1:10)
 * 2) CDKN2A 7C (1:10)
 * 3) MMP12 31B (1:10)
 * 4) THPO 33A (1:10)

--> Templates, use 2 μL
 * 1) KAH126-1, 6/15/10
 * 2) KAH126-1 +dox, 6/15/10
 * 3) KAH132-8, 6/15/10
 * 4) KAH132-8 +dox, 6/15/10
 * 5) KAH154-2, 6/15/10
 * 6) KAH154-2 +dox, 6/15/10
 * 7) FTRx, 6/15/10
 * 8) FTRx +dox, 6/15/10

--> 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


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