User:Karmella Haynes/Notebook/Polycomb project/2010/10/12

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10/12/10

 * &#x2713; ChIP qPCR: trouble shooting
 * &#x2713; Order oligos: new qPCR primer pairs
 * &#x2713; Western: ChIP/ co-IP optimization

ChIP qPCR troble shooting
 * Low C(t) values: primers too dilute; 750 nM = 30 μL of 10 μM primer mix in 400 μL total volume (not 500 μL total volume!)
 * GAPDH B is more efficient than experimental primers. Check GC content and Tm's of primers. Use Primer 3 to pick better primer pairs

Western: ChIP co-IP > Optimization IP for αH3K27me3 > IP samples from 10/08/10; 60 μL each, ready to load > Prep input 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. 30 min.

> Block: 5% milk/PBST, 4°C/ > 1 hr.

> Primary staining: 5% BSA/PBST, 4°C/o.n.
 * 1) rabbit α-DsRed 632496, 1:1000, 2.5 mL
 * 2) rabbit α-H3K27me3 07-449, 1:1000, 2.5 mL

10/13/10 > 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
 * KAH126-1: 43 kD
 * H3K27me3: 15 - 17 kD

Conclusions: IP is not specific to antibody. Next time, try ChIP protocol from Casey


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