User:Karmella Haynes/Notebook/Polycomb project/2010/10/08

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10/08/10

 * &#x2713; ChIP qPCR: 126-1 & 132-8 continued
 * &#x2713; ChIP qPCR: primer test for new primer pairs

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) INKARF G
 * 2) MMP12 A3
 * 3) MMP12 B
 * 4) TNF A

--> 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

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):
 * KAH126-1 Input, pos (1:1)
 * 0 template (dH2O)
 * 1) MMP12 C2
 * 2) MMP12 C3
 * 3) MMP12 D1
 * 4) MMP12 D2
 * 5) TNF C2
 * 6) TNF C3
 * 7) TNF D1
 * 8) TNF D2

--> 750 nM primer mix = 30 μL 10 μM mix + 470 μL H2O

--> 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):

> Conclusions:
 * Use MMP12 C2, TNF C3 for future experiments (TNF C3 overlaps with TNF C1)
 * Design new primers for MMP12 and TNF D regions


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