User:Karmella Haynes/Notebook/Polycomb project/2011/03/10

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03/10/11

  • ✓ RNA prep/ cDNA: KAH126 -/+ dox
  • ✓ Luc activity assay: luc repressed 6-day, Pc-TF 4-day



RNA prep
> Use TriZOL
> Dissolve each pellet in 15 μL THE RNA Storage Sln.

Sample A260 260/280 ng/ul uL = 2ug RNA dH2O
1. KAH126-1, no dox 4.284 1.88 171.3 do not use* ---
2. KAH126-1, no dox 8.057 1.98 322.3 6.2 1.8
3. KAH126-1, no dox 16.255 2.01 650.2 3.1 4.9
4. KAH126-1, 4-day 9.217 1.96 368.7 5.4 2.6
5. KAH126-1, 4-day 6.483 2.01 259.3 7.7 0.3
6. KAH126-1, 4-day 21.578 1.93 863.1 2.3 5.7

Note: *Accidentally added phenol:chloroform:isoamyl alcohol to cells (sample #1)


> oligo(dT) Primer annealing
--> Set up four reactions for each condition:

  1. Dox-: use sample #3
  2. Dox+: use sample #6
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
--> 16 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 0.8 μL RNase H, 37°C/ 20 min.
--> Store at -20°C



Luc Activity Assay

Luc activity assay > Cells: dox-induced Gal4-EED expression/ 2 days, followed by transfection for 4 days
1-6. KAH160/MV1 (human Pc-TF), 0, 50, 100, 200, 400, 800 ng plasmid
7-12. KAH170/MV1 (human Pc-TF), 0, 50, 100, 200, 400, 800 ng plasmid
> Sample processing:

  • Add 500 μL medium to cells in rep 1 set (measured before); Resuspend cells in 1 mL medium total
  • Aliquot 3x 100 μL samples into 96-well plate
  • Save the rest for cell count (Millipore Scepter)
  • Normalize each value to cell count



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