User:Karmella Haynes/Notebook/Polycomb project/2010/10/05

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10/05/10

 * &#x2713; ChIP qPCR: optimize primers

ChIP qPCR > Optimization to make sure input gives low C(t) compared to no template ctrl. > Set up each reaction in triplicate > Templates (use 4.0 μL, 39 rxns each): > Primers (6 rxns each): --> 750 nM primer mix = 30 μL 10 μM mix + 470 μL H2O
 * KAH126-1 Input, pos (1:1)
 * 0 template (dH2O)
 * 1) INKARF D
 * 2) INKARF E
 * 3) INKARF F
 * 4) INKARF G
 * 5) MMP12 A
 * 6) MMP12 A 2 (new)
 * 7) MMP12 A 3 (new)
 * 8) MMP12 B
 * 9) MMP12 C
 * 10) TNF A (new)
 * 11) TNF B (new)
 * 12) TNF C (new)
 * 13) GAPDH B

--> Aliquot 31.5 primer mix into 1st well of each triplicate set --> Add 13.5 DNA 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/ 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

Results (0 = signal below threshold):

> Design and order new MMP12 (downstream of tss), MMP12 C, and TNF A primers > Continue with ChIP qPCR for good primers


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