User:Karmella Haynes/Notebook/Polycomb project/2010/12/06

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12/06/10

 * &#x2713; ChIP qPCR 1: H3K27me3 IP set; optimize template load
 * &#x2713; ChIP qPCR 2: H3K27me3 IP set; INKARF primers
 * &#x2713; In vitro H3 tail binding assay: order H3K9me3 peptide and streptavidin beads
 * &#x2713; Cell culture: thaw KAH126-1, 128-8.3, 129-4, 130-4 for histone peptide binding assays
 * &#x2713; HepG2 TREx: change medium to DMEM plain (blast slowing cell growth?)

ChIP qPCR 1 > Set up each reaction in triplicate > Templates (use 1, 2, 4.5 μL): Note: Accidentally switched fx input and K27 > Primers (57 rxns total): --> Plate 1
 * FTRx dx input, pos (9 wells)
 * FTRx dx H3K27me3 IP, uk (9)
 * FTRx dx myc, neg (9)
 * FTRx fx input, pos (9) *
 * FTRx fx H3K27me3 IP, uk (9) *
 * FTRx fx myc, neg (9)
 * 0 template (3)
 * 1) GAPDH B2

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

--> Aliquot 31.5 primer mix into 1st well of each triplicate --> Add 13.5 (4.5 x 3) DNA+dH2O 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

> Conclusion: GAPDH all volumes give C(t) = 27 for all input samples. 2 μL gives C(t) of about 33 - 35 for others. Melt curve peaks = 88.0 for all samples. Peak heights below threshold for K27me3 and myc IP as expected. Use 2 μL for future experimental samples.

ChIP qPCR 2 > Set up each reaction in triplicate > Templates (use 2.0 μL): > Primers (21 rxns pre primer pair): --> Plate 2 --> 750 nM primer mix = 3 μL 100 μM each primer + 394 μL H2O
 * FTRx dx input, pos
 * FTRx dx H3K27me3 IP, uk
 * FTRx dx myc, neg
 * FTRx fx input, pos
 * FTRx fx H3K27me3 IP, uk
 * FTRx fx myc, neg
 * 0 template
 * 1) INKARF D1
 * 2) INKARF E2
 * 3) INKARF F1
 * 4) INKARF G3

--> Aliquot 39.0 primer mix into 1st well of each triplicate --> Add 6.0 (2.0 x 3) DNA to 39.0 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


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