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