User:Karmella Haynes/Notebook/miR Trigger/2009/12/24

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

12/24/09

  • ✓ RNA prep & cDNA for KAH93, 94, 95, 108/MV2 transfected Dox+/- JDS33-47 cells
  • ✓ Split U2OS plain: 1x 1:10 flask; 2x 6-well, 2.5x105/well, ab-free
  • ✓ Split JDS33-47: 1x 1:10 flask

RNA prep & cDNA
> 6-well plates: use 1 mL TRIZol per well
> Dissolve pellet in 15 μL THE RNA Storage Solution

> Measure RNA concentrations

Sample A260 260/280 ng/μL vol = 2 μg
1. KAH93/MV2 7.657 1.98 306.9 6.5
2. KAH93/MV2 (Dox) 0.060 6.73 2.4 x
3. KAH94/MV2 10.346 1.97 413.8 4.8
4. KAH94/MV2 (Dox) 0.517 1.82 20.7 x
5. KAH95/MV2 18.231 1.88 729.3 2.7
6. KAH95/MV2 (Dox) 15.123 1.92 604.9 3.3
7. KAH108/MV2 26.328 1.98 1053.1 1.9
8. KAH108/MV2 (Dox) 23.386 2.00 935.4 2.1
9. no DNA 23.601 2.01 944.1 2.1
10. no DNA (Dox) 17.490 2.00 699.6 2.9

> oligo(dT) Primer annealing
--> Discard low yeild samples (2, 4); retry later --> 2 rxns for KAH108, 108/Dox, no DNA, no DNA/ Dox (12 total)

Reagent Vol
total RNA up to 5 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

Reagent Vol Mix (x12)
10x RT buffer 2.0 24.0
25 mM MgCl2 4.0 48.0
0.1 M DDT 2.0 24.0
RNaseOUT 1.0 12.0
SuperScript III RT 1.0 12.0
  10.0 μL 120.0 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