User:Karmella Haynes/Notebook/Polycomb project/2010/06/18

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06/18/10

  • ✓ Western: re-probe 1.2, 1.3, 1.4, 2.1 blots
  • ✓ RT-PCR (set #2): 126-1, 126-3, 132-8, 154-2, FTRx -/+ dox, 4 days RNA preps (from 6-well); cDNA



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 29.222 2.01 1168.9 4.5 μL (5 μg)
2. KAH126-1 dox 20.197 2.01 807.9 6.0 μL (5 μg)
3. KAH126-3 76.472 1.78 3058.9 1.5 μL (5 μg)
4. KAH126-3 dox 28.070 2.00 1122.8 4.5 μL (5 μg)
5. KAH132-8 39.697 1.98 1587.9 3.0 μL (5 μg)
6. KAH132-8 dox 8.744 1.99 349.8 8.0 μL (2.8 μg)
7. KAH154-2 62.768 1.92 2510.7 2.0 μL (5 μg)
8. KAH154-2 dox 25.490 2.01 1019.6 5.0 μL (5 μg)
9. FTRx 41.747 2.00 1669.9 3.0 μL (5 μg)
10. FTRx dox 40.600 1.99 1624.0 3.0 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


Western
> Block w/ 5% BSA/PBST (filter sterilized) at R.T./ 1 hour
> Primaries, use 0.5% BSA/ PBST:
1.2. rabbit α-H3K27me3 07-449, 1:500, 3 mL
1.3. mouse α-H3K27me3 ab6002, 1:500, 3 mL
1.4. rabbit α-H3K4 C42D8, 1:500, 3 mL
2.1. mouse α-p16 ab16880, 1:100, 3 mL


6/19/10
--> Secondaries (R.T./ 1 hr.; 0.5% BSA/PBST):
1.3, 2.1. donkey α-mouse IgG-HRP, 1:5000, 5 mL
1.2, 1.4. donkey α-rabbit IgG-HRP, 1:5000, 5 mL

Image:KAH 061710 WB1.tif

> Conclusions:



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