User:Karmella Haynes/Notebook/Polycomb project/2010/07/07

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07/07/10

 * &#x2713; Senescence assay: Pc-ATF lines and FTRx +rotenone
 * &#x2713; RT-PCR (set #4): RNA prep/ cDNA synth.

Senescence assay > Samples: 1,2. KAH126-1 -/+ dox 3,4. KAH126-3 -/+ dox 5,6. KAH132-8 -/+ dox 7,8. KAH154-2 -/+ dox 9,10. FTRX -/+ 0.2 μg/mL rotenone

> Treat w/ 1.5 μg/mL C12-FDG/ 37°C/ 45 min. before harvesting for flow cytometry > Wash once with DPBS and resuspend in 0.5 mL DPBS

RNA Preps/ cDNA synth.

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

--> Make 1/10 dilutions in THE RNA storage solution --> Note: RT-PCR signal was not good when cDNA was made with 5 μg RNA (see 7/02/10). Successful RT-PCR of p16INK was seen with cDNA synthesized from ~1 μg of RNA (see 5/03/10). This time use less RNA (1 μg). Store remaining RNA prep @ -80°C.

> 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


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