The dual HR and NHEJ reporter assay was performed as previously described (Arnoult et al., 2017 (link)). Briefly, indicated cells were plated in a 6-well plates at a 50% confluence one day before transfection. Cells were transfected with 500ng of pCBA I-SceI plasmid (Addgene), 500ng of pLCN-DRR plasmid (Addgene) plasmid, and 500ng of pCAGGS-DRR-mCherry-Donor-EF1a-BFP plasmid (Addgene). After 72 hr incubation cells were collected and BFP, mCherry, and eGFP expression was detected by using the Becton-Dickinson LSR18 machine and analyzed with FlowJo version 7 software module.
Pcba 1 scei plasmid
Manufactured by Addgene
The PCBA-I-SceI plasmid is a laboratory tool designed for molecular biology applications. It contains the I-SceI endonuclease gene, which can be used to introduce site-specific double-strand breaks in DNA sequences.
Automatically generated - may contain errors
4 protocols using pcba 1 scei plasmid
1
Dual HR and NHEJ Reporter Assay
2
Measuring DNA Repair Pathways in U2OS Cells
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For the DR-GFP assays, HR-U2OS cells (Luessing et al. 2021, under review) were used, while for the NHEJ (EJ5) U2OS derivative cell line was kindly provided by J. Stark. 2 × 106 cells were transfected with 5 ug pCBA-I-SceI plasmid (Addgene #26477), 40 nmol siRNA and 1 ug Cerulean-c1 plasmid (Addgene #54604) using electroporation (BioRad electroporator). Cells were harvested and resuspended in 500 ul PBS containing 40 nM TOPRO-3 iodide (Life Technologies, #T3605) to identify live cells. The cells were gated for live cells, doublet exclusion and transfected cells (cerulean-positive). A minimum of 20 000 transfected cells were then assessed for the expression of GFP. FACS analysis was carried out using BD FACSCANTOII and BD-FACS DIVA software. The remaining cells were used for checking the knock-down efficiency by western blotting.
3
Assessing DNA Repair Pathways in U2OS Cells
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Cells were transfected using siRNA as described above. For the DR-GFP assay, our own U2OS stable cell line was used (HR-U2OS). For the NHEJ (EJ5) and MMEJ (EJ2) assays U2OS derivative cell lines were kindly provided by J. Stark ( Bennardo et al., 2008 Bennardo N. Cheng A. Huang N. Stark J.M.
Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 2008; 4: e1000110 Crossref PubMed Scopus (670) Google Scholar
). 2x10 6 cells were transfected with 5ug pCBA-I-SceI plasmid (Addgene #26477), 40nmol siRNA and 1ug cerulean-c1 plasmid (Addgene #54604) using electroporation (BioRad electroporator). 24h post transfection, cells were split into a 6-well dish and grown for a further 24h. Then cells were harvested by trypsinisation and resuspended in 500ul PBS containing 40nM TOPRO-3 iodide (Life Technologies, #T3605) to identify live cells. The cells were gated for live cells and transfected cells (ceruleanpositive). Doublet were excluded. A minimum of 20,000 transfected cells were then assessed for GFP expression. FACS analysis was carried out using BD FACSCANTOII and BD-FACS DIVA software. Remaining cells were used for western blotting to determine knock-down efficiency.
Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 2008; 4: e1000110 Crossref PubMed Scopus (670) Google Scholar
). 2x10 6 cells were transfected with 5ug pCBA-I-SceI plasmid (Addgene #26477), 40nmol siRNA and 1ug cerulean-c1 plasmid (Addgene #54604) using electroporation (BioRad electroporator). 24h post transfection, cells were split into a 6-well dish and grown for a further 24h. Then cells were harvested by trypsinisation and resuspended in 500ul PBS containing 40nM TOPRO-3 iodide (Life Technologies, #T3605) to identify live cells. The cells were gated for live cells and transfected cells (ceruleanpositive). Doublet were excluded. A minimum of 20,000 transfected cells were then assessed for GFP expression. FACS analysis was carried out using BD FACSCANTOII and BD-FACS DIVA software. Remaining cells were used for western blotting to determine knock-down efficiency.
4
DNA Repair Assays in U2OS Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the DR-GFP assays, HR-U2OS cells (Luessing et al 2021, under review) were used, while for the NHEJ (EJ5) U2OS derivative cell line was kindly provided by J. Stark 76 . 2x10 6 cells were transfected with 5ug pCBA-I-SceI plasmid (Addgene #26477), 40nmol siRNA and 1ug Cerulean-c1 plasmid (Addgene #54604) using electroporation (BioRad electroporator). Cells were harvested and resuspended in 500ul PBS containing 40nM TOPRO-3 iodide (Life Technologies, #T3605) to identify live cells. The cells were gated for live cells, doublet exclusion and transfected cells (ceruleanpositive). A minimum of 20.000 transfected cells were then assessed for the expression of GFP.
FACS analysis was carried out using BD FACSCANTOII and BD-FACS DIVA software. The remaining cells were used for checking the knock-down efficiency by Western Blotting.
FACS analysis was carried out using BD FACSCANTOII and BD-FACS DIVA software. The remaining cells were used for checking the knock-down efficiency by Western Blotting.
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