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