Human Pluripotent Stem Cell Gene Targeting
Responsibilities of the Investigator
- Discuss experimental design with facility directors before beginning your project.
- Provide a map of the construct including size, regions of homology, selection strategy, enzyme sites and oligo binding sites.
- Identify a unique restriction site to linearize your plasmid. Provide gel documentation of your test digest.
- Provide 100ug of purified plasmid at 1ug/ul.
- Develop a reliable genotyping assay (long PCR, Southern, or both) before submission of DNA to the facility.
- Screen picked clones for targeted candidates within one month (during which time the clones are stored at -80o).
- Confirm genotype of candidate clones by Southern blot.
- Submit a Request Form in iLabs to initiate the project.
Services Provided by the Facility
- The gene-targeting facility will linearize your plasmid, electroporate into ES/iPS cells, perform selection and pick clones.
- ES cell lines provided:
- iPS cell lines provided:
- We guarantee two rounds of electroporations (if necessary) for each targeting construct.
- Picked clones will be stored at -80o for up to one month, during which time the investigator must screen for candidate clones.
- The facility will expand candidate clones for freezing and confirmation of genotyping by Southern blot (investigator provides Southern blot confirmation).
- After confirmation of homologous recombination, the facility will karyotype up to four of these clones. Any additional expansion and/or karyotyping of clones will be subject to an additional charge.
Nov 04, 2019