User:Karmella Haynes/Notebook/Polycomb project/2010/07/08

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

07/08/10

  • ✓ RT-PCR: try p16INK 7B (p16INK f2/r2) on various cDNA samples
  • ✓ Western: protein preps from 10 cm and 6-well plates, rotenone -/+ FTRx cells

--> Also do RNA prep/ cDNA SYNTH. for 6-well samples (for p16INK RT-PCR)

  • ✓ Transfection: puromycin selection plates (25 mL) for KAH130, 131 (dox- samples, 100k and 50k cells), and "no DNA" neg. control (100k cells)
  • ✓ Cell culture: split confluent plates 1:10
  • ✓ Senesence assay: long term experiment -/+ dox; 6-well plates for KAH126-1, KAH126-3, KAH132-8, KAH154-2, KAH128-8.3, KAH129-4
  • Cytology (prep): 12-well plate for KAH126-1, KAH126-3, KAH132-8, KAH154-2, KAH128-8.3, KAH129-4; DAPI vs. RFP (images for paper) Don't use regular culture plates, use glass-bottom 24-well (#P24G-1.5-13-F) instead
  • ✓ Order primers: p16INK 7C (f3/r3); EZH2 (f1/r1); p14ARF 6B (f2/r2); ATF3 (f1/r1); CCND2 (f1/r1)



RT-PCR
--> cDNA templates

Sample RNA used to make cDNA
1,2. EZH2 kd 4-day (11/03/09) 1:1, 1:10 (?)
3,4. EZH2 kd #2 8-day (9/18/09) 1:1, 1:10 (?)
5,6. KAH126-1 +dox 4-day (2/21/10) 1:1, 1:10 0.41 μg
7,8. FTRx no dox 4-day (2/21/10) 1:1, 1:10 2.0 μg (?)
9,10. KAH126-1 +dox (6/15/10) 1:1, 1:10 5.0 μg
11,12. KAH126-1 +dox (6/18/10) 1:1, 1:10 5.0 μg
13,14. KAH126-1 +dox (6/23/10) 1:1, 1:10 5.0 μg
15,16. KAH126-1 +dox (7/07/10) 1:1, 1:10 1.0 μg

--> Primer pair: p16INK4a(7B) (make fresh)
--> See 4/29/10 for successful p16INK RT-PCR


Reagent 1:1 cDNA MM (x10) 1:10 cDNA MM (x10) File:KAH 070810 gel1.tif
15 μL/lane; 2% agarose
cDNA 0.5 (1:1) --- 5.0 (1:100) ---
10μM primers 1.0 10.0 1.0 10.0
2x GoTaq Green 10.0 100.0 10.0 100.0
dH2O 8.5 85.0 3.5 35.0
  20.0 μL 20.0 μL
 
 
 
 
 
 
 
 

--> Aliquot 19.5 μL ("1:1" tubes) or 15.0 μL ("1:10" tubes) of each DNA mix to appro. wells
--> Add 0.5 (1:1) or 5.0 (1:100) 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/ ∞


--> Conclusion: Reducing amount of RNA for cDNA synth. appears not to improve specificity of product. DNA contamination? Order new primers based on Agger et al. 2009, where one primer is designed to overlap exon junction (maybe more specific). Also order primers for mRNA's of genes included in ChIP set.



Western: rotenone-treated FTRx

> Protein preps (use RIPA + 1x p.i. cocktail):

  1. 10 cm plate FTRx +DMSO, 500 μL RIPA
  2. 10 cm plate FTRx +0.2 μg/mL rotenone, 500 μL RIPA
  3. 6-well (1 well) FTRx +DMSO, 200 μL RIPA
  4. 6-well (1 well) FTRx +0.2 μg/mL rotenone, 200 μL RIPA

--> Rotate @ 4°C/ 1 hour
--> Spin @ 15,000rpm/ 4°C/ 15 min. Save supernantant @ -20°C until gels arrive. --> Continue with Western 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 μg)
1. FTRx -rotenone 52.704 1.97 2108.2 0.9 μL (2 μg)
2. FTRx +rotenone 13.185 2.04 527.4 3.8 μL (2 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 (x2)
10x RT buffer 2.0 4.0
25 mM MgCl2 4.0 8.0
0.1 M DDT 2.0 4.0
RNaseOUT 1.0 2.0
SuperScript III RT 1.0 2.0
  10.0 μL 20.0 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