User:Karmella Haynes/Notebook/Polycomb project/2010/06/10

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


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06/10/10

 * &#x2713; ChIP: 126-1 and U2OS plain samples (corrected protocol)
 * &#x2713; PCR: test ChIP primers on U2OS plain quality control DNA

ChIP > Do 4 IP's for each sample > Samples: > Antibodies for IP
 * 1) 126-1, ~1.7 mL/ IP
 * 2) U2OS plain (remainder from last attempt), ~1.2 mL/ IP
 * 1) mouse α-PolII 8WG16 (5 μg)
 * 2) rabbit α-myc ab9106 (5 μg)
 * 3) rabbit α-H3K27me3 07-449 (5 μg)
 * 4) mouse α-IgG sc-2025 (2 μg) + rabbit α-IgG sc-2027 (2 μg)

PCR > Test ChIP PCR primers (expected amplicons in bp) > Templates
 * 1) INKARF A f1/r1 = 125
 * 2) INKARF B f1/r1 = 134
 * 3) INKARF C f1/r1 = 138
 * 4) INKARF D f1/r1 = 135
 * 5) INKARF E f1/r1 = 86
 * 6) INKARF F f1/r1 = 100
 * 7) INKARF G f1/r1 = 115
 * 8) INKARF H f1/r1 = 113
 * 9) INKARF I f1/r1 = 110
 * 10) ATF3 A f1/r1 = ?
 * 11) CCND2 A f1/r1 = ?
 * 12) GAPDH A f1/r1 = 117
 * 13) GAPDH B f1/r1 = 128
 * 1) U2OS sonicated DNA quality control sample (from 5/11/2010)
 * 2) Flp-in T-REx genomic DNA

--> Aliquot 19.0 μL of each master mix to appro. wells --> Add 1.0 primers to each well --> PCR (96-well)
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
 * 72°C/ 3 min.
 * 4°C/ ∞

--> Success! All primer pairs work. DNA from sonicated chromatin can be amplified by all primer pairs.


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