U2OS and HEK293 cells were cultured in Advanced DMEM (Life Technologies) supplemented with 10% FBS, 2 mM GlutaMax (Life Technologies), and penicillin/streptomycin at 37 °C with 5% CO2. U2OS cells (program DN-100) and HEK293 cells (program CM-137) were transfected in 20 μl Solution SE on a Lonza Nucleofector 4-D according to the manufacturer’s instructions. dsODN integration rates were assessed by restriction fragment length polymorphism (RFLP) assay using NdeI. Cleavage products were run and quantified by a Qiaxcel capillary electrophoresis instrument (Qiagen) as previously described32 (link).
Protocol detail
Transfection and RFLP Analysis
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Assay
Capillary electrophoresis
Cells
Cleavage
Hek293 cells
Penicillin
Restriction fragment length polymorphism
Streptomycin
Corresponding Organization : Massachusetts General Hospital
Other organizations : Center for Cancer Research
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Variable analysis
independent variables
- Transfection of U2OS cells (program DN-100)
- Transfection of HEK293 cells (program CM-137)
- DsODN integration rates
- Culture of U2OS and HEK293 cells in Advanced DMEM supplemented with 10% FBS, 2 mM GlutaMax, and penicillin/streptomycin at 37 °C with 5% CO2
- Restriction fragment length polymorphism (RFLP) assay using NdeI to assess dsODN integration rates
- Quantification of cleavage products by Qiaxcel capillary electrophoresis instrument
- No positive or negative controls were explicitly mentioned in the input protocol.
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