Materials and Reagents

1. U2OS or RG37 cell lines
   Note: U2OS are osteosarcoma cells with active p53 pathway, RG37 are SV40-immortalized human fibroblasts with inactive p53 pathway. There are cells harbouring the substrate designed to measure Homology Directed repair. However, any cell line in which the HDR substrate has been inserted can be used.
   
2. Culture medium (serum-free medium)
   
3. siRNA for gene of interest (for example rad51, its depletion decreases the efficiency of repair of a DNA DSB by homologous recombination)
   
4. Plasmid pcDNA3myc-NLS-I-SceI (coding for I-SceI created by Puget et al. (2005))
   
5. INTERFERin Transfection Reagent (Ozyme, catalog number: POL409-10 )
   
6. JetPEI Transfection Reagent (Ozyme, catalog number: POL101-10 )
   
7. Trypsin
   
8. Glycerol
   
9. SDS
   
10. Beta-mercaptoethanol
    
11. Bromophenol blue
    
12. Laemmli buffer (see Recipes)
    
13. Phosphate Buffered Saline (PBS) (see Recipes)
    

Equipment

1. 35 mm tissue culture plate
   
2. Tissue culture set up (e.g. 37 °C, 5% CO₂ incubator)
   
3. Flow cytometer
   
4. Western blotting apparatus
   

Procedure