User:Karmella Haynes/Notebook/Polycomb project/2011/04/08

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04/08/11 ✓ cDNA synthesis: Pc-TF RNA samples from 4/07/11 cDNA Synthesis Sample A260 260/280 ng/ul uL = 2ug RNA dH2O 1. KAH129-4 26.686 2.01 1067.4 2.0 6.0 (4 rxns) 2. " 34.923 1.97 1396.9 1.5 6.5 3. " 33.629 2.02 1345.2 --- --- 4. KAH129-4 +dox 9.354 1.97 374.2 5.0 3.0 (2 rxns) 5. " 9.824 1.97 393.0 5.0 3.0 (2 rxns) 6. " 8.259 2.00 330.3 --- --- 7. KAH154-2 28.441 2.02 1137.6 2.0 6.0 8. " 32.997 2.02 1319.9 1.5 6.5 9. " 29.262 2.02 1170.5 --- --- 10. KAH154-24 +dox 9.206 1.98 368.2 --- --- 11. " 12.520 2.04 500.8 4.0 4.0 12. " 10.814 1.97 432.6 5.0 3.0 13. KAH156-5 37.510 2.01 1500.4 1.5 6.5 14. " 5.759 1.84 230.4 --- --- 15. " 40.389 1.99 1615.5 1.5 6.5 16. KAH156-5 +dox 34.323 2.02 1372.9 1.5 6.5 17. " 25.292 2.01 1011.7 --- --- 18. " 31.572 1.98 1262.9 1.5 6.5 19. KAH157-1 21.889 2.00 875.5 2.5 5.5 20. " 88.213 1.47 3528.5 --- --- 21. " 34.218 1.98 1368.7 1.5 6.5 22. KAH157-1 +dox 30.727 2.02 1229.1 1.5 6.5 23. " 25.637 2.03 1025.5 2.0 6.0 24. " 19.326 1.94 773.1 --- --- > oligo(dT) Primer annealing --> Do 4 rxn's for 129-4 #1 --> 2 rxn's ea. for 129-4 +dox #4 & #5 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 --> 16 reactions total Reagent Vol Mix (x21) 10x RT buffer 2.0 42.0 25 mM MgCl2 4.0 84.0 0.1 M DDT 2.0 42.0 RNaseOUT 1.0 21.0 SuperScript III RT 1.0 21.0   10.0 μL --> Add 10 μL mix to each annealing rxn. --> 50°C/ 50 min., 80°C/ 5 min., ice --> Add 0.8 μL RNase H, 37°C/ 20 min. --> Store at -20°C