User:Karmella Haynes/Notebook/Polycomb project/2010/02/20

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02/20/10

  • ✓ RNA/ cDNA prep: 4-day dox Pc-ATF cultures (plate set A)
  • ✓ Pc-ATF RT-PCR cultures: split plate set B into two duplicates; start new plate for KAH130-2
  • Cytology: seed 24-well cover slip plate for H3K27me/ H3K9me staining Very sparse cell growth. Do over on Tuesday
  • ✓ Cultures: Split confluent Pc-ATF lines 1:10 (10 cm plates)



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 2.347 1.98 93.9 8.0 μL (0.75 μg)
2. KAH126-1 dox 1.295 1.94 51.8 8.0 μL (0.41 μg)
3. KAH127-4 2.722 2.01 108.9 8.0 μL (0.87 μg)
4. KAH127-4 dox 0.723 2.17 28.9 8.0 μL (0.23 μg)
5. KAH128-8 11.440 1.91 457.6 4.4 μL (2.00 μg)
6. KAH128-8 dox 9.511 1.94 380.4 5.2 μL (2.00 μg)
7. KAH129-4 24.166 2.01 966.6 2.1 μL (2.00 μg)
8. KAH129-4 dox 0.885 2.00 35.4 8.0 μL (0.28 μg)
9. KAH130-2 1.617 2.01 64.7 8.0 μL (0.52 μg)
10. KAH130-2 dox 83.459 1.61 3338.4 0.6 μL (2.00 μg)
11. KAH131-9 33.028 2.00 1321.1 1.5 μL (2.00 μg)
12. KAH131-9 dox 0.328 2.31 13.1 8.0 μL (0.11 μg)
13. KAH132-8 2.941 1.70 117.6 8.0 μL (0.94 μg)
14. KAH132-8 dox 4.776 1.98 191.0 8.0 μL (1.53 μg)
15. KAH133-1 15.005 1.97 600.2 3.3 μL (2.0 μg)
16. KAH133-1 dox 37.503 2.00 1500.1 1.3 μL (2.0 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
--> 8 reactions total

Reagent Vol Mix (x16)
10x RT buffer 2.0 32.0
25 mM MgCl2 4.0 64.0
0.1 M DDT 2.0 32.0
RNaseOUT 1.0 16.0
SuperScript III RT 1.0 16.0
  10.0 μL 160.0 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