Assay Overview

Plate Format: 96 well
Plate Coating: Poly-L-Lysine

Assay Cell Culture

HCT116

Transgene: Mad2+/-
Origin: Swanton Lab
Cell Medium: E4+10%FCS+1%PenStrep
Cell Seeding Density Per Well: 4000

Delivery Protocol - Reverse/Wet transfection

Protocol: Mix siRNA in 10uL HBSS to 37.5nM final concentration. Mix 0.3uL Lipofectamine RNAiMAX in 10uL OPTIMEM. Mix together on plate and leave for 20 minutes. Add cells in 80ul/well. Culture the cells for further 72hours
Delivery Reagent: Lipofectamine RNAiMAX
Delivery Reagent Cat No: 13778500
Delivery Reagent Supplier: Invitrogen
Delivery Reagent Concentration: 0.3ul/well
Silencing Reagent Concentration: 37.5nM
Silencing Reagent Supplier: Dharmacon
Silencing Reagent Origin: HTS Lab

Cell Fixation

Fixed in 80% Ethanol overnight
Ethanol
80%
long-term at -20C

Assay Staining

phospho H2AX staining
3%BSA/PBS
1hour
DAPI
1ug/ml
1 hour

Assay Readouts