User:Karmella Haynes/Notebook/Polycomb project/2010/09/21

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

09/21/10

  • ✓ RT-PCR 1: EZH2 kd set, EZH2 primers
  • ✓ RT-PCR 2: EZH2 kd "8-day 2" pair, re-do all primers using 2x/ 4x more nt template
  • ✓ ChIP: sonicate KAH126-1 & 132-8
  • ✓ Microscopy: 158-3 cells showed no indication of polycomb bodies. Put this expt. on hold.



RT-PCR 1
> Repeat EZH2 22A RT-PCR from 9/20/10
--> Use 1:10 and 1:100 dilutions of cDNA

File:KAH 092110 gel1.tif

> Conclusions: Slight indication of EZH2 kd (need to quantitate using ImageJ). Need to re-do 8-day kd 2 because of underloading for the nt sample.



RT-PCR2
> EZH kd set
--> Templates

  1. n.t. kd 2 8-day, 9/18/09 (2x)
  2. EZH2 kd 2 8-day, 9/18/09
  3. n.t. kd 2 8-day, 9/18/09 (4x)
  4. EZH2 kd 2 8-day, 9/18/09

--> Primers

  1. p16INK 7C (94 bp) (4 rxns)
  2. MMP12 31A (127 bp) (4 rxns)
  3. THPO 33A (103 bp) (4 rxns)
  4. TNF 34A (119 bp) (4 rxns)
  5. GAPDH 21A (100 bp), 1:1000 cDNA (4 rxns)
  6. EZH2 22A (71 bp), 1:10 cDNA (4 rxns)


Reagent Single rxn
cDNA 2.0 total vol
10 μM primers 1.0
2x GoTaq Green 10.0
dH2O 6.5
  20.0 μL

--> Use 0.5 cDNA + 1.5 dH2O for EZH2 kd
--> Use 1.0 cDNA + 1.0 dH2O for nt kd 2x
--> Use 2.0 cDNA for nt kd 4x

--> PCR (96-well)

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
  • 72°C/ 3 min.
  • 4°C/ ∞


File:KAH 092110 gel2.tif

> Conclusions:

  • 2x nt for GAPDH probably misloaded (accidentally added 1:1 cDNA to this well, rinsed out, and replaced with 1:1000, but there might be carry-over)
  • Repeat GAPDH RT-PCR (9/23/10)

File:KAH 092310 gel1.tif


> Conclusions: This RT-PCR confirms that 2x nt was previously mis-loaded. Use this 4x nt/ EZH2 kd pair for ImageJ quantitation.