User:Karmella Haynes/Notebook/Polycomb project/2010/12/30

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


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12/30/10

 * &#x2713; ChIP qPCR: 128-8.3 and 129-4

ChIP qPCR > Set up each reaction 4x > Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL): > Primers (24 rxns per primer pair): --> Plate 1
 * 128-8 (40) input, pos
 * 128-8 (41) myc IP, uk
 * 128-8 (42) IgG IP, neg
 * 129-4 (43) input, pos
 * 129-4 (44) myc IP, uk
 * 129-4 (45) IgG IP, neg
 * 1) GAPDH A3
 * 2) GAPDH B2
 * 3) INKARF D1
 * 4) INKARF E2

--> Plate 2
 * 1) INKARF F1
 * 2) INKARF G3
 * 3) MMP12 A3
 * 4) MMP12 B2

--> Plate 3
 * 1) MMP12 C2
 * 2) MMP12 D3
 * 3) TNF A2
 * 4) TNF B2

--> Plate 4
 * 1) TNF C4
 * 2) TNF D3

--> 750 nM primer mix = 3 μL each 100 μM primer + 394 μL H2O

--> Aliquot 52.0 primer mix into 1st well of each 4x set --> Add 8.0 (2.0 x4) DNA to 52.0 primer mix --> Aliquot 15.0 rxn mix to other 3 wells in each 4x 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, 57°C/ 15 sec, 72°C/ 15 sec] x45
 * Melt curve range 57°C -> 95°C/ 0.5°C per step


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