User:Karmella Haynes/Notebook/Polycomb project/2010/10/03

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10/03/10

 * &#x2713; ChIP qPCR: experimental sample set 126-1
 * &#x2713; Western: ChIP/ Co-IP for 126-1 (DsRed and H3K27me3)
 * &#x2713; Culture: split HepG2's 1:10

qPCR > Set up each reaction in triplicate > Templates (use 4.5 μL): > Primers (12 rxns each): --> 750 nM primer mix = 30 μL 10 μM mix + 470 μL H2O
 * KAH126-1 Input, pos (1:25)
 * KAH126-1 αmyc IP, exp (1:25)
 * KAH126-1 αIgG IP, neg (1:25)
 * 0 template (dH2O)
 * INKARF D
 * INKARF E
 * INKARF F
 * INKARF G
 * MMP12 A
 * MMP12 B
 * MMP12 C
 * GAPDH B

--> Aliquot 31.5 primer mix into 1st well of each triplicate set --> Add 12.5 1:25 template DNA to primer mix --> Aliquot 15.0 rxn mix to other 2 wells in 3x 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/ 3°C per step

> Conclusions:
 * C(t) still not very far from background. Try again using 2.0 μL 1:1 template

Western blot: ChIP/ Co-IP > IP samples from 10/01/10; 30 μL each, ready to load > Prep input and sup samples: 22.5 protein + 7.5 4x loading dye --> 4x loading dye (Invitrogen) w/ freshly added DTT (200 mM final) > Use 10-well gel (loading volume = 25 μL) > Electroblot: 1 hr. 15 min.

> Block: 5% milk/PBST, 4°C/overnight

10/04/10 > Primary staining: 5% BSA/PBST, 4°C/o.n.
 * 1) rabbit α-DsRed 632496, 1:1000, 5 mL
 * 2) rabbit α-H3K27me3 07-449, 1:500, 5 mL

10/05/10 > Secondary staining: 5% milk/PBST, R.T./ 1 hr. > Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html for KAH proteins)
 * 1) donkey α-rabbit-HRP, 1:5000; 5 mL
 * 2) donkey α-rabbit-HRP, mouse α-GAPDH-HRP 1:5000; 5 mL
 * KAH126-1: 43 kD
 * H3K27me3: 15 - 17 kD

Conclusions:


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