User:Karmella Haynes/Notebook/Polycomb project/2010/10/07

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10/07/10

 * &#x2713; ChIP qPCR: 126-1 and 132-8 samples
 * &#x2713; ChIP optimization: second step (5-hour binding)

ChIP qPCR > 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, 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 (#16), pos
 * 6) KAH132-8 αmyc IP (#18), unk
 * 7) KAH132-8 αIgG IP (#20), neg
 * 8) 0 template (dH2O)
 * 1) INKARF D
 * 2) INKARF E
 * 3) INKARF F
 * 4) GAPDH B

--> 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 optimization > Continued from yesterday > Make enough complete sonication buffer for bead-ab wash and washes for elution (12 mL) > Chromatin + antibody samples (1,2) --> Add 10 μL prepped Protein A Sepharose beads --> Rotate at 4°C/ 5 hours > Beads + antibody samples --> Wash off unbound antibody: pellet beads (3000 rpm/ 4°C/ 3 min.), discard sup, wash w/ complete sonication buffer (keep on ice); repeat 2x --> Resuspend in 250 μL complete sonication buffer + 250 μL chromatin (KAH126-1 dx) --> Rotate at 4°C/ 5 hours

> Wash and elute --> Same protocol as αmyc-bead conjugate IP, but gently wash 3x instead of 4x --> Elute with 60 μL 1x loading dye + 50 mM DDT (enough to load two Western wells) --> Heat at 65°C/ 10 min., vortex every 2 min. --> Pellet beads, transfer supernatant to new tube --> Heat supernatant at 100°C/ 5 min. --> Store at -20°C until Western blot.


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