User:Karmella Haynes/Notebook/Polycomb project/2011/04/26

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04/26/11

 * &#x2713; ChIP qPCR: plates ch6, ch7, ch8, ch9

ChIP qPCR > NPPA and EOMES showed H3K27me3 enrichment; check for Pc-TF enrichment > Set up each reaction 4x

>Templates --> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL):
 * 1) 132-8 (21) input, pos
 * 2) 132-8 (36) myc IP, uk
 * 3) 132-8 (37) IgG IP, neg
 * 4) 126-1 (16) input, pos
 * 5) 126-1 (32) myc IP, uk
 * 6) 126-1 (33) IgG IP, neg
 * 7) 128-8.3 (40) input, pos
 * 8) 128-8.3 (41) myc IP, uk
 * 9) 128-8.3 (42) IgG IP, neg
 * 10) 129-4 (43) input, pos
 * 11) 129-4 (44) myc IP, uk
 * 12) 129-4 (45) IgG IP, neg

> Plate ch6 primers --> 750 nM primers (48 rxns per primer pair):
 * 1) EOMES A1
 * 2) EOMES B2

> Plate ch7 primers --> 750 nM primers (48 rxns per primer pair):
 * 1) EOMES C3
 * 2) EOMES D3

> Plate ch8 primers --> 750 nM primers (48 rxns per primer pair):
 * 1) NPPA B2
 * 2) NPPA C2

> Plate ch9 primers --> 750 nM primers (48 rxns per primer pair):
 * 1) NPPA A1
 * 2) NPPA D1

--> 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) --> Note: Use increased annealing temp compared to previous ChIP PCR's (new primers optimized for 58°C) --> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film
 * 95°C/ 5 min.
 * [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x45
 * Melt curve range 58°C -> 95°C/ 0.5°C per step


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