User:Karmella Haynes/Notebook/miR Trigger/2009/12/31

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12/31/09

  • ✓ RNA prep/ cDNA synth.: KAH93, 94, 95/MV2 transfected cells (+/- Dox)



RNA prep & cDNA
> 6-well plates: use 1 mL TRIZol per well
> Dissolve pellet in 10 μL THE RNA Storage Solution

> Measure RNA concentrations

Sample A260 260/280 ng/μL vol = 2 μg
1. KAH93/MV2 8.866 1.98 354.7 5.6 μL
2. KAH93/MV2 (Dox) 10.554 1.96 422.2 4.7
3. KAH94/MV2 16.045 1.90 641.8 3.1
4. KAH94/MV2 (Dox) 15.813 1.90 632.5 3.2
5. KAH95/MV2 15.973 1.95 638.9 3.1
6. KAH95/MV2 (Dox) 41.982 1.97 1679.3 1.2

> oligo(dT) Primer annealing
--> details

Reagent Vol
total RNA (up to 5μg) 2 μg
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
--> 6 reactions total

Reagent Vol Mix (x12)
10x RT buffer 2.0 12.0
25 mM MgCl2 4.0 24.0
0.1 M DDT 2.0 12.0
RNaseOUT 1.0 6.0
SuperScript III RT 1.0 6.0
  10.0 μL 60.0 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