User:Karmella Haynes/Notebook/Polycomb project/2010/10/04

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10/04/10

 * &#x2713; ChIP qPCR: KAH126-1
 * &#x2713; Western blot: continued from 10/03/10

ChIP qPCR > Repeat from 10/04/10 using more template DNA > Set up each reaction in triplicate > Templates (use 2.0 μL, 8 rxns each): > Primers (12 rxns each): --> 750 nM primer mix = 30 μL 10 μM mix + 470 μL H2O
 * KAH126-1 Input, pos (1:1)
 * KAH126-1 αmyc IP, exp (1:1)
 * KAH126-1 αIgG IP, neg (1:25)
 * 0 template (dH2O)
 * INKARF D
 * INKARF E
 * INKARF F
 * INKARF G
 * MMP12 A
 * MMP12 B
 * MMP12 C
 * GAPDH B

--> Aliquot 31.5 primer mix into 1st well of each triplicate set --> Add 13.5 DNA mix to primer mix --> Aliquot 15.0 rxn mix to other 2 wells in 3x set

Bio-Rad CFX96 qPCR (Kirschner lab) --> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film
 * 95°C/ 10 min.
 * [95°C/ 10 sec, 58°C/ 10 sec, 72°C/ 10 sec] x45
 * Melt curve: 95°C/ 10 sec, 57°C -> 95°C/ 0.5 °C per step

> Conclusions: Results not as clear as previous. Try original PCR conditions and 4.5 uL template for primer optimization of full set (INKARF, MMP12, TNF)


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