User:Karmella Haynes/Notebook/PcTF Genomics/2015/01/28

Pc-TF Genomics

Main project page Previous entry      Next entry

01/28/15 Cytology of KAH126 Cytology of KAH126 Last attempt at looking for Pc speckles in KAH-126 was at Harvard: https://www.openwetware.org/wiki/User:Karmella_Haynes/Notebook/Polycomb_project/2010/04/04 Try again at different concentrations of dox induction (maybe over-expression gives high background?) Cell plating (1/23/15) Poly-L-lysine coating of cells (done last week) - follow Mammalian IFC protocol Seed cells at 100k per well (aliquot in 1mL volumes into 24-well plate with poly-lysine coated cover slips on bottom) Same day - Induce cells with 1.0, 0.1, and 0.01 μg/mL dox; final volume = 2 mL Tube 1: Added 8 μL 1 mg/mL dox to 4 mL growth medium (2x target concentration) Tube 2: 0.5 mL from tube 1 into 4.5 mL medium (") Tube 3: 0.5 mL from tube 2 into 4.5 mL medium (") Added 1 mL tube 1 into 1 mL cells in wells 1 & 2 (dilution by 1/2 --> reached target concentration of dox) Added 1 mL tube 2 into 1 mL cells in wells 3 & 4 (") Added 1 mL tube 3 into 1 mL cells in wells 5 & 6 (") Wells 7 & 8 got 1 mL medium without dox Expression - next day (1/24/15) No signs of PC speckles Expression - 5 days post induction (1/28/15) Neglected the cells, checked today out of curiosity; growth medium still healthy color A few cells show what look like Pc speckles in the nucleus; most others have diffuse RFP Fix and permeabilize these for IF staining - follow Mammalian IFC protocol