User:Karmella Haynes/Notebook/Polycomb project/2010/02/08

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

02/08/10

  • ✓ RNA preps/ cDNA synth.: Polycomb-ATF lines (dox-induced, ~2 days)
  • ✓ HEK293 #4 Luc: 1x 1:10 flask, 1x 24-well plate (~10,000 cells per well) for Gal4-EED transfection/ luc assay
  • ✓ U20S Flp-in T-REx: 1x 1:10 flask, 1x 6-well (for Pc-ATF gene expression control)



RNA Preps/ cDNA synth.


> RNA Preps
--> Use 1 mL TriZol/ well (6-well dish)
--> Combine duplicate after resuspending RNA pellet in 6 μL THE RNA Storage Solution

Sample OD 260 260/280 ng/μL vol = 2 μg
1. KAH126-1 10.051 2.01 402.1 4.5 μL
2. KAH127-4 6.877 1.87 275.1 7.5 μL
3. KAH128-8 52.594 1.72 676.3 3.0 μL
4. KAH129-4 7.789 1.94 311.6 6.5 μL
5. KAH130-2 8.398 1.95 335.9 6.0 μL
6. KAH131-9 6.813 1.81 173.0 8.0 μL
7. KAH132-8 51.697 1.76 923.0 2.0 μL
8. KAH133-1 5.709 1.84 228.4 8.0 μL


> oligo(dT) Primer annealing

Reagent Vol
total RNA (2 μg) 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
--> 8 reactions total

Reagent Vol Mix (x8)
10x RT buffer 2.0 16.0
25 mM MgCl2 4.0 32.0
0.1 M DDT 2.0 16.0
RNaseOUT 1.0 8.0
SuperScript III RT 1.0 8.0
  10.0 μL 80.0 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


Personal tools