Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
(030619)
takarabio.com
Takara Bio USA, Inc.
Page 3 of 24
Table of Figures
Figure 1. Schematic of CRISPR/Cas9-based editing to create gene knockouts and knockins ............................................... 5
Figure 2. Using DEF-CS technology to generate edited clonal cell lines ............................................................................... 6
Figure 3. Workflow for gene editing of hiPS cells using components of the Cellartis iPSC rCas9 Electroporation and
Single-Cell Cloning System .................................................................................................................................................... 9
Figure 4. Workflow for generating sgRNA using components of the Cellartis iPSC rCas9 Electroporation and Single-Cell
Cloning System ..................................................................................................................................................................... 10
Figure 5. Designing a forward PCR primer to generate a DNA template for an sgRNA containing your target sequence . 11
Figure 6. Gel electrophoresis of the PCR product ................................................................................................................ 12
Figure 7. sgRNA yield over time .......................................................................................................................................... 13
Figure 8. Workflow for electroporation-based delivery of rCas9/sgRNA complexes .......................................................... 15
Figure 9. Recommended density of starting culture used for single-cell cloning ................................................................. 20
Figure 10. A single cell seeded in one well generates an emerging colony .......................................................................... 22
Figure 11. Clonal colonies, ready for transfer to larger wells and scale-up .......................................................................... 23
Table of Tables
Table I. Examples of sgRNA concentration and yield using different elution volumes. ...................................................... 14
Table II. Preparation of coating solution for a 48-well plate. ............................................................................................... 16
Table III. Preparation of DEF-CS medium for dissociation and recovery. ........................................................................... 16
Table IV. Workflow for single-cell cloning and expansion using the Cellartis iPSC rCas9 Electroporation and Single-Cell
Cloning System. .................................................................................................................................................................... 19
Table V. Preparation of coating solution for a 96-well plate. ............................................................................................... 20
Table VI. Preparation of medium for dissociation and single-cell seeding. ......................................................................... 21
Table VII. Preparation of coating solution for a 48-well plate. ............................................................................................ 23
