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

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09/21/10

 * &#x2713; RT-PCR 1: EZH2 kd set, EZH2 primers
 * &#x2713; RT-PCR 2: EZH2 kd "8-day 2" pair, re-do all primers using 2x/ 4x more nt template
 * &#x2713; ChIP: sonicate KAH126-1 & 132-8
 * &#x2713; 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



> 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 --> Primers
 * 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
 * 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)

--> 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/ ∞



> 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)



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


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