User:Karmella Haynes/Notebook/Polycomb project/2010/10/11

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


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10/11/10

 * &#x2713; ChIP qPCR: 126-1, 132-8 continued
 * &#x2713; HepG2 culture: Split HepG2 for transfections; back-up stock 1:5
 * &#x2713; HEK Gal4EED culture: induce 12-well plate with dox (set 1); back-up stock 1:5

ChIP qPCR > Set up each reaction in triplicate > Templates (use 4.0 μL, 12 rxns each): > Primers (24 rxns each): --> 750 nM primer mix = 30 μL 10 μM mix + 470 μL H2O
 * 1) KAH126-1 Input (#16), pos
 * 2) KAH126-1 αmyc IP (#18), unk
 * 3) KAH126-1 αIgG IP (#20), neg
 * 4) 0 template (dH2O)
 * 5) KAH132-8 Input (#21), pos
 * 6) KAH132-8 αmyc IP (#23), unk
 * 7) KAH132-8 αIgG IP (#25), neg
 * 8) 0 template (dH2O)
 * 1) MMP12 C2
 * 2) TNF B
 * 3) TNF C3
 * 4) TNF C1

--> 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


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