User:Karmella Haynes/Notebook/Polycomb project/2010/06/23

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06/23/10 ✓ RT-PCR (set #3): 126-1, 126-3, 132-8, 154-2, FTRx -/+ dox, 4 days RNA preps (from 6-well); cDNA ✓ Cell culture: thaw lines 129-3, 129-4 (2/11/10), 129-4 (1/23/10), 131-6, 131-9 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. KAH126-1 44.714 1.99 1788.6 3.0 μL (5 μg) 2. KAH126-1 dox 25.641 2.00 1025.7 5.0 μL (5 μg) 3. KAH126-3 47.346 1.98 1893.8 2.5 μL (5 μg) 4. KAH126-3 dox 33.672 2.00 1346.9 4.0 μL (5 μg) 5. KAH132-8 48.205 1.98 1928.2 2.5 μL (5 μg) 6. KAH132-8 dox 3.715 2.02 148.6 8.0 μL (1.2 μg) 7. KAH154-2 28.685 2.00 1147.4 4.5 μL (5 μg) 8. KAH154-2 dox 32.468 2.00 1298.7 4.0 μL (5 μg) 9. FTRx 41.192 1.99 1647.7 3.0 μL (5 μg) 10. FTRx dox 47.743 1.98 1909.7 2.5 μL (5 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 --> 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