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

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07/07/10

  • ✓ Senescence assay: Pc-ATF lines and FTRx +rotenone
  • ✓ 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

Sample OD 260 260/280 ng/μL Vol. to use for cDNA synth. (1 μg)
1. KAH126-1 43.867 2.00 1754.7 5.7 μL 1/10 dil. (1 μg)
2. KAH126-1 dox 20.959 2.02 838.4 1.2 μL, undiluted (1 μg)
3. KAH126-3 44.811 2.00 1792.5 5.6 μL 1/10 dil. (1 μg)
4. KAH126-3 dox 34.679 2.01 1387.2 7.2 μL 1/10 dil. (1 μg)
5. KAH132-8 51.953 1.99 2078.1 4.8 μL 1/10 dil. (1 μg)
6. KAH132-8 dox 12.590 2.05 503.6 2.0 μL, undiluted (1 μg)
7. KAH154-2 45.047 2.00 1801.9 5.5 μL 1/10 dil. (1 μg)
8. KAH154-2 dox 40.504 2.02 1620.1 6.2 μL 1/10 dil. (1 μg)
9. FTRx 53.656 1.97 2146.3 4.6 μL 1/10 dil. (1 μg)
10. FTRx dox 49.389 2.00 1975.6 5.1 μL 1/10 dil. (1 μg)

--> 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

Reagent Vol
total RNA (up to 2μg) up to 8 μL
50μM oligo(dT) primer 1.0
10 mM dNTP mix 1.0
DEPC-treated water ---
  10.0 μL --> 65°C/ 5 min.; ice/ 1 min.


> cDNA synthesis mix
--> 10 reactions total

Reagent Vol Mix (x10)
10x RT buffer 2.0 20.0
25 mM MgCl2 4.0 40.0
0.1 M DDT 2.0 20.0
RNaseOUT 1.0 10.0
SuperScript III RT 1.0 10.0
  10.0 μL 100.0 μL

--> 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