User:Karmella Haynes/Notebook/Polycomb project/2010/03/02

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03/02/10

  • ✓ RNA prep/ cDNA: 130-2 (4 day dox) samples
  • ✓ RT-PCR (continue tomorrow)


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. KAH130-2 7.757 1.95 310.3 6.4 μL (2.00 μg)
2. KAH130-2 dox 7.913 1.97 316.5 6.3 μL (2.00 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
--> 2 reactions total

Reagent Vol Mix (x3)
10x RT buffer 2.0 6.0
25 mM MgCl2 4.0 12.0
0.1 M DDT 2.0 6.0
RNaseOUT 1.0 3.0
SuperScript III RT 1.0 3.0
  10.0 μL 30.0 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


RT-PCR

> Reaction 1: Loading optimization
--> 4-day dox induction set, plus EZH2 k.d. (8 day) and nt (8 day)
--> Use GAPDH primers and 1:10,000 cDNA template dilutions
--> Note: EZH2 kd (8 day) (dilute 3x)


Reagent Volume Master mix (x22) RT-PCR optimization 3/02/10
15 μL/lane; 1% agarose 		Adjustment for RT-PCR:
1. EZH2 k.d. = 1/2
2. nt = 2x
3. 126-1 = 1x
4. 126-1(dox) = 1/2
5. 127-4 = 2x
6. 127-4(dox) = 2x
7. 128-8 = 1/2
8. 128-8(dox) = 1x
9. 129-4 = 1x
10. 129-4(dox) = 1x
11. 130-2 = 2x
12. 130-2(dox) = 2x
13. 131-9 = 2x
14. 131-9(dox) = 2x
15. 132-8 = 1/2
16. 132-8(dox) = 1x
17. 133-1 = 1x
18. 133-1(dox) = 1x
19. FTRx = 1/2
20. FTRx(dox) = 1x
cDNA 0.5 ---
10μM primers 1.0 22.0
2x GoTaq Green 10.0 220.0
dH2O 8.5 187.0
  20.0 μL
 
 
 
 
 
 
 
 

--> Aliquot 19.5 μL of each DNA mix to appro. wells
--> Add 0.5 cDNA to each well
--> PCR (96-well)

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
  • 72°C/ 3 min.
  • 4°C/ ∞


> Reaction 2: gene activation assay

--> Primers/ cDNA dilution:

  1. MYT1 (1D)/ 1:1
  2. MYT1 (1G)/ 1:1
  3. p15INK (5)/ 1:1
  4. p16INK (7B)/ 1:1
  5. RUNX3 (8A)/ 1:1
  6. DUSP4 (15A)/ 1:1
  7. TGFB1 (10A)/ 1:100
  8. PLAU (12A)/ 1:100
  9. KRT17 (14A)/ 1:100
  10. LAMB3 (16A)/ 1:100
  11. PPPIR (17A)/ 1:100
  12. GAPDH (21A)/ 1:10,000


Reagent Volume 1:1 DNA mix (x7) 1:100 DNA mix (x6) 1:10,000 DNA Primer mix (x21)
cDNA 0.5 3.5 x adjustment 3.0 x adjustment 0.5 x adjustment ---
10μM primers 1.0 --- --- --- 22.0
2x GoTaq Green 10.0 --- --- --- 220.0
dH2O 8.5 63.0 - cDNA 54.0 - cDNA 9.0 - cDNA ---
  20.0 μL

--> Aliquot 9.0 μL of each DNA mix to appro. wells
--> Aliquot 11.0 μL of each primer mix to appro. wells
--> PCR (96-well)

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
  • 72°C/ 3 min.
  • 4°C/ ∞

RT-PCR 3/02/10


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