Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
(030619)
takarabio.com
Takara Bio USA, Inc.
Page 2 of 24
Table of Contents
I.
Introduction ..................................................................................................................................................................... 4
II. List of Components ......................................................................................................................................................... 6
III. Additional Materials Not Supplied ................................................................................................................................. 7
A. Required ...................................................................................................................................................................... 7
B. Recommended ............................................................................................................................................................. 7
IV.
General Considerations ............................................................................................................................................... 8
A. Storage and Handling .................................................................................................................................................. 8
B. Transferring Human iPS Cells to the DEF-CS Culture System .................................................................................. 8
V. Complete Experimental Workflow ................................................................................................................................. 9
VI.
In Vitro
Transcription of an sgRNA Containing the Desired Target Sequence .......................................................... 9
A. Protocol Overview ...................................................................................................................................................... 9
B. Generating the DNA Template ................................................................................................................................. 10
C. Performing the
In Vitro
Transcription (IVT) Reaction ............................................................................................. 12
D. Purifying the Transcribed sgRNA ............................................................................................................................. 14
VII.
Editing hiPS Cells by Electroporation ...................................................................................................................... 15
A. Protocol Overview .................................................................................................................................................... 15
B. Preparing Media and Cells ........................................................................................................................................ 15
C. Preparing the rCas9/sgRNA RNP complex .............................................................................................................. 17
D. Electroporating Cells Using the Neon Transfection System ..................................................................................... 18
VIII. Single-Cell Cloning of Edited Cells .......................................................................................................................... 19
A. Protocol Overview .................................................................................................................................................... 19
B. Single-Cell Seeding into a 96-Well Plate.................................................................................................................. 20
C. Culturing Single-Cell Colonies ................................................................................................................................. 22
IX.
Passaging Cells from the 96-Well Plate to a 48-Well Plate ...................................................................................... 23
A. Coating a 48-Well Plate ............................................................................................................................................ 23
B. Preparing DEF-CS SCC Medium for Passaging ....................................................................................................... 23
C. Passaging ................................................................................................................................................................... 23
X. Scaling up from the 48-Well Plate ................................................................................................................................ 24
XI.
References ................................................................................................................................................................. 24
