User:Karmella Haynes/Notebook/Polycomb project/2010/10/17

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10/17/10

 * &#x2713; ChIP qPCR: continue primer test

ChIP qPCR primer test > Optimization to make sure input gives low C(t) compared to no template ctrl. > Set up each reaction in triplicate > Templates (use 4.5 μL): > Primers (6 rxns each):
 * KAH132-8 Input, pos (1:1)
 * 0 template (dH2O)
 * 1) GAPDH C2
 * 2) MMP12 A3
 * 3) MMP12 B1
 * 4) MMP12 B2
 * 5) MMP12 B3
 * 6) MMP12 C2
 * 7) MMP12 C4
 * 8) MMP12 D3
 * 9) MMP12 D4
 * 10) TNF A1
 * 11) TNF A2
 * 12) TNF B1
 * 13) TNF B2
 * 14) TNF C1
 * 15) TNF C4
 * 16) TNF D3

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

--> Make primer mix in 1st well of each set of six wells (two triplicates) --> Aliquot 31.5 primer mix into 1st well second triplicate --> Add 13.5 (4.5 x 3) DNA to 31.5 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, 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):

> Conclusions: Use primers with C(t) of 27-30
 * INKARF: D1, E2, F1, G3
 * GAPDH: A3, B2, C1
 * MMP12: A3, B2, C2, D3
 * TNF: A2, B2, C4, D3


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