Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
(030619)
takarabio.com
Takara Bio USA, Inc.
Page 17 of 24
NOTE:
For electroporation, each sample requires 1 x 10
5
cells. However, due to the potential variation of
pipette
and tip volumes, we recommend preparing 1.5X the necessary volume of cell suspension (i.e., 1.5 x 10
5
cells) for electroporation with a 10-µl Neon Tip to ensure that there is sufficient volume.
1.
Aspirate the media from the starting culture vessel and wash the cell layer once with D-PBS –/–.
2.
Add TrypLE Select Enzyme (1X) to the initial culture vessel (see Table III) and incubate for 5 min at
37°C, or until the cell layer has detached. Detachment can be aided by swirling the cell culture flask
or by tapping the side of the cell culture flask firmly but gently.
3.
Resuspend the cells in DEF-CS medium (see Table III) and pipet up and down several times to ensure
a single-cell suspension. (The cells will aggregate if left too long in TrypLE enzyme.)
4.
Take an aliquot of the cell suspension and measure the cell density using your preferred method.
5.
Centrifuge the cells at 400
g
for 5 min in a 15-ml conical tube.
6.
Wash the cells once with D-PBS –/–, then resuspend cells in Buffer R (included with Neon kits) at a
concentration of 2 x 10
7
cells/ml (i.e., 1.5 x 10
5
cells in 7.5 µl).
7.
Keep the cell suspension on ice until use.
C.
Preparing the rCas9/sgRNA RNP complex
Next, rCas9 and sgRNA components are combined to form RNP complexes for electroporation.
1.
Thaw Guide-it Recombinant Cas9 (Electroporation-Ready) and sgRNA solutions at room temperature.
NOTE:
We recommend preparing aliquots upon initial thawing of Guide-it Recombinant Cas9
(Electroporation-Ready) to avoid repeated freeze/thaw cycles.
2.
Combine the following components in a 200-µl PCR tube to mix the rCas9 protein and sgRNA at a
5:1 mass ratio. The molar ratio of rCas9 protein to sgRNA will be approximately 1:1 in this mixture,
and the total volume will be 7.5 µl. Be sure to use the same buffer that was used to resuspend the
cells.
NOTE:
The reaction volume indicated below is 1.5X the required volume.
Per reaction:
0.45 μl* sgRNA (e.g., 1 µg/µl)
0.75 μl
*
Guide-it Recombinant Cas9 (Electroporation Ready) (3 µg/µl)
6.3 μl* Resuspension Buffer R or T
7.5 μl
*
Total volume
*The added volume of sgRNA will vary depending on sgRNA concentration, and the added volume of
Resuspension Buffer should be adjusted such that the total reaction volume is 7.5 µl. The volumes
indicated above are based on a sgRNA concentration of 1 µg/µl.
NOTES:
•
Make a master mix if you are performing multiple electroporations.
•
To maximize electroporation efficiency, the combined volume of the rCas9 and sgRNA solutions
should be ≤20% of the total volume of the rCas9/sgRNA RNP complex reaction (e.g., for the 7.5-
µl reaction specified above, the combined volume of the sgRNA and rCas9 solutions should be
≤1.5 µl).
•
If you plan to use donor DNA to induce HDR-mediated knockin, add the DNA
after
the
subsequent incubation step (Step 3). We recommend using ≤1 µg of DNA for knockin
experiments. Adjust the volume of Resuspension Buffer R or T included in the reaction such
that the final volume upon addition of donor DNA is 7.5 µl.
