User:Karmella Haynes/Notebook/Polycomb project/2010/10/16

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10/16/10

 * &#x2713; ChIP qPCR: test primers
 * &#x2713; HepG2 Pc-ATF transfections: expand 1/2 transfected cells to 2.0 μg/mL puromycin 10 cm plates
 * &#x2713; HEK Gal4EED induction: split cells into new plates (1:2), keep old plate, refresh dox, add dox to 1-day samples; assay tomorrow

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 4.0 μL, 39 rxns each): > Primers (6 rxns each):
 * KAH132-8 Input, pos (1:1)
 * 0 template (dH2O)
 * 1) INKARF D1
 * 2) INKARF D2
 * 3) INKARF D3
 * 4) INKARF D4
 * 5) INKARF E1
 * 6) INKARF E2
 * 7) INKARF F1
 * 8) INKARF F2
 * 9) INKARF G1
 * 10) INKARF G2
 * 11) INKARF G3
 * 12) GAPDH A2
 * 13) GAPDH A3
 * 14) GAPDH B1
 * 15) GAPDH B2
 * 16) GAPDH C1

--> 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: Looks like using proper primer concentration improved the C(t) values. Use the following for qPCR (C(t) = 27 - 30) > Continue primer test tomorrow


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