User:Karmella Haynes/Notebook/Polycomb project/2010/08/26

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>      </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

08/26/10

  • ✓ Microscopy: immunostaining (overnight)
  • ✓ RT-PCR: p16 RT-PCR



Microscopy
> Immunostaining: see 8/16/10 for optimization expt.
> Cells: KAH130-4, 158-4, 131-4, 159-2
> Antibodies:

  1. Histone H3 ab1791 (rabbit)
  2. H3K27me3 07-449 (rabbit)
  3. H3K9me3 9754 (rabbit)
  4. H3K4me3 9751 (rabbit)
  5. Bmi1 ab14389 (mouse)
  6. PolII 8WG16 (mouse)



RT-PCR
> See 8/17/10 for primer optimization expt.
> Primer pairs:

  1. p16INK 7C (1:1, no dilution)
  2. GAPDH 21A (1:1000 dilution)

RT-PCR
> cDNA from RNeasy RNA preps
1,2. KAH126-1 -/+ dox
3,4. KAH128-8.3 -/+ dox
5,6. KAH129-4 -/+ dox
7,8. KAH132-8 -/+ dox
9,10. KAH154-2 -/+ dox
11,12. KAH156-5 -/+ dox
13,14. KAH157-1 -/+ dox
15,16. KAH158-4 -/+ dox
17,18. KAH159-1 -/+ dox
19,20. FTRx -/+ dox


Reagent 1:1 cDNA MM (x22) File:KAH 082610 PCR1.tif
15 μL/lane; 2% agarose
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 (1:1 or 1:1000) cDNA to each well
--> 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/ ∞

(* Last time used 35 cycles; this time use 32 to reduce background amplification)