User:Karmella Haynes/Notebook/Polycomb project/2010/03/19

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03/19/10

  • ✓ cDNA: 8-day dox induction Pc-ATF cell line set
  • ✓ Cell cultures: Refresh medium for KAH126, 130, 132 starter cultures (20% FBS)

RNA prep/ cDNA synth.


> RNA Preps (yesterday)
--> Frozen: 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. KAH126-1 9.123 1.97 364.9 6.0 μL (~2 μg)
2. KAH126-1 dox 3.008 2.01 120.3 8.0 μL (0.962 μg)
3. KAH127-4 9.104 1.92 364.2 6.0 μL (~2 μg)
4. KAH127-4 dox 0.961 2.01 38.4 8.0 μL (0.307 μg)
5. KAH128-8 59.256 1.94 2370.2 1.0 μL (~2 μg)
6. KAH128-8 dox 64.348 1.85 2573.9 1.0 μL (~2 μg)
7. KAH129-4 6.818 1.88 272.7 8.0 μL (~2 μg)
8. KAH129-4 dox 1.221 1.98 48.9 8.0 μL (0.391 μg)
9. KAH130-2 33.903 1.98 1356.1 2.0 μL (~2 μg)
10. KAH130-2 dox 7.925 1.89 317.0 7.0 μL (~2 μg)
11. KAH131-9 22.391 2.02 895.6 3.0 μL (~2 μg)
12. KAH131-9 dox 27.345 2.00 1093.8 2.0 μL (~2 μg)
13. KAH132-8 54.492 1.96 2179.7 1.0 μL (~2 μg)
14. KAH132-8 dox 0.500 2.20 20.0 8.0 μL (0.160 μg)
15. KAH133-1 17.600 2.04 704.0 3.0 μL (~2 μg)
16. KAH133-1 dox 20.825 2.04 833.0 3.0 μL (~2 μg)
17. ctrl 89.407 1.36 3576.3 0.6 μL (~2 μg)
18. ctrl dox 53.633 1.98 2145.3 1.0 μL (~2 μg)


> 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
--> 8 reactions total

Reagent Vol Mix (x16)
10x RT buffer 2.0 36.0
25 mM MgCl2 4.0 52.0
0.1 M DDT 2.0 36.0
RNaseOUT 1.0 18.0
SuperScript III RT 1.0 18.0
  10.0 μL 180.0 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



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