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