User:Karmella Haynes/Notebook/Polycomb project/2010/10/02

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10/02/10

 * &#x2713; ChIP qPCR optimization: Optimization PCR

qPCR > Optimization > Set up each reaction in triplicate > Templates (use 1.0, 2.0, 4.0, and 0 μL): --> Make master mix of 13x for each template at each volume; 8 "mixes" total > Primers: --> 750 nM primer mix = 30 μL 10 μM mix + 470 μL H2O
 * KAH126-1 Input, positive (1:50)
 * KAH126-1 αIgG IP, negative (1:50)
 * INKARF D (24 rxns)
 * INKARF E (24 rxns)
 * INKARF F (24 rxns)
 * GAPDH B (24 rxns)

Bio-Rad CFX96 qPCR (Kirschner lab) --> Use Bio-Rad 96-well low profile plate MLL-9601 + clear 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/ 3°C per step

> Conclusions:
 * ~1 C(t) increase per doubling of template amount. Average C(t) for 4 μL template reactions (Input pos ctrl) is about 26 - 36 (a little too weak, neg ctrl is ~40). Use 4.5 μL of 1:25 dilution for future reactions.
 * Separate pipetting of template into primer mix yeilds too much variation btwn tech replicates. Mix 3 rxns in one tube (45 μL), then aliquot 15 μL.


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