User:Karmella Haynes/Notebook/Polycomb project/2011/04/21

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

mm/dd/yy

 * &#x2713; Luc assay: HEK-luc rep +Pc-TF transfections (plate 2A)
 * &#x2713; ChIP qPCR: Plates ch4

ChIP qPCR > First check for enrichment of H3K27me3 at putative target genes > Set up each reaction 4x

>Plate ch4 --> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL): --> 750 nM primers (12 rxns per primer pair):
 * FTRx (29) input, pos
 * FTRx (38) H3K27me3 IP, uk
 * FTRx (39) IgG IP, neg
 * 1) NPPA A1
 * 2) NPPA B2
 * 3) NPPA C2
 * 4) NPPA D1
 * 5) EOMES A1
 * 6) EOMES B2
 * 7) EOMES C3
 * 8) EOMES D3

--> 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


 * }