User:Karmella Haynes/Notebook/Polycomb project/2010/06/18

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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06/18/10

 * &#x2713; Western: re-probe 1.2, 1.3, 1.4, 2.1 blots
 * &#x2713; RT-PCR (set #2): 126-1, 126-3, 132-8, 154-2, FTRx -/+ dox, 4 days RNA preps (from 6-well); cDNA

RNA Preps/ cDNA synth.

> RNA Preps --> Use 1 mL TriZol/ well (6-well dishes) --> Resuspend RNA pellet in 10 μL THE RNA Storage Solution

> oligo(dT) Primer annealing

> cDNA synthesis mix --> 10 reactions total

--> Add 10 μL mix to each annealing rxn. --> 50°C/ 50 min., 80°C/ 5 min., ice --> Add 1.0 μL RNase H, 37°C/ 20 min. --> Store at -20°C

Western > Block w/ 5% BSA/PBST (filter sterilized) at R.T./ 1 hour > Primaries, use 0.5% BSA/ PBST: 1.2. rabbit α-H3K27me3 07-449, 1:500, 3 mL 1.3. mouse α-H3K27me3 ab6002, 1:500, 3 mL 1.4. rabbit α-H3K4 C42D8, 1:500, 3 mL 2.1. mouse α-p16 ab16880, 1:100, 3 mL

6/19/10 --> Secondaries (R.T./ 1 hr.; 0.5% BSA/PBST): 1.3, 2.1. donkey α-mouse IgG-HRP, 1:5000, 5 mL 1.2, 1.4. donkey α-rabbit IgG-HRP, 1:5000, 5 mL



> Conclusions:


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