User:Karmella Haynes/Notebook/Polycomb project/2010/12/20

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


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12/20/10

 * &#x2713; ChIP: DNA purification day 3 (etOH ppt; resuspend in 500 10 mM Tris pH 8.0)
 * &#x2713; ChIP qPCR: new IP set and previous input samples
 * &#x2713; ChIP: myc/IgG IP's for 128-8.3 and 129-4 chromatin
 * &#x2713; Cell culture: expand thawed cultures 126-3, 154-2, 132-8, FTRx; split 126-1
 * &#x2713; HepG2 TREx: transfer from 6-well to 24-well plate to increase cell density (split into 2 wells)

ChIP qPCR > Set up each reaction 4x > Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL): > Primers (48 rxns pre primer pair): --> Plate 2 --> 750 nM primer mix = 3 μL each 100 μM primer + 394 μL H2O
 * 126-1 (16) input, pos
 * 126-1 (32) myc IP, uk
 * 126-1 (33) IgG IP, neg
 * 130-4 (06) input, pos
 * 130-4 (34) myc IP, uk
 * 130-4 (35) IgG IP, neg
 * 132-8 (21) input, pos
 * 132-8 (36) myc IP, uk
 * 132-8 (37) IgG IP, neg
 * FTRx (29) input, pos
 * FTRx (38) H3K27me3 IP, uk
 * FTRx (39) IgG IP, neg
 * 1) INKARF D1
 * 2) INKARF E2
 * 3) INKARF F1
 * 4) INKARF G3

--> Aliquot 52.0 primer mix into 1st well of each 4x set --> Add 8.0 (2.0 x4) DNA to 52.0 primer mix --> Aliquot 15.0 rxn mix to other 3 wells in each 4x 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: 128-8.3, 129-4 > Samples (all form. x-linked chromatin) 1/2. 128-8: + 30 μL αmyc-beads (3400); + 30 μL mouse IgG-beads (3420) 3/4. 129-4: " > Rotate @ 4°C/ overnight


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