User:Karmella Haynes/Notebook/Polycomb project/2011/04/17

{| 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"|

04/17/11

 * &#x2713; ChIP qPCR: plate ch1

ChIP qPCR > See 12/20/11 > Set up each reaction 4x

>Plate ch1 --> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL): --> 750 nM primers (24 rxns per primer pair):
 * 126-1 (16) input, pos
 * 126-1 (32) myc IP, uk
 * 126-1 (33) IgG IP, neg
 * 132-8 (21) input, pos
 * 132-8 (36) myc IP, uk
 * 132-8 (37) IgG IP, neg
 * 1) ATOH1 A1
 * 2) ATOH1 B3
 * 3) ATOH1 C2
 * 4) ATOH1 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


 * }