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. 2016 Nov 15;1:13. [Version 1] doi: 10.12688/wellcomeopenres.10011.1

Figure 7. Simple targeting pipeline for generation of genetically modified LUHMES cell lines using CRISPR technology.

Figure 7.

Cells are Nucleofected with the plasmids and ssODNs necessary for the specific targeting experiment and taken for FACS sorting two days later. After approximately 10 days single cell colonies will have expanded enough to require dissociation and transfer to 24-well plates. From 24-well plates cells can be frozen down for storage in liquid nitrogen and some cells harvested for genomic DNA. The first step of the screening strategy is to perform an RFLP assay to identify a subset of clones that will be taken for genomic DNA sequencing which will identify cell lines that are positive for a clean KI. For more detailed information see methods and materials.