Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
(030619)
takarabio.com
Takara Bio USA, Inc.
Page 14 of 24
D.
Purifying the Transcribed sgRNA
For use with the Guide-it IVT RNA Clean-Up Kit.
NOTE:
Before purifying your sgRNA, prepare the IVT Wash Buffer by adding 24 ml of 96–100%
ethanol.
1.
Add 78 µl of RNase Free Water to the reaction mixture (from Step C) for a total volume of 100 µl.
Transfer all 100 µl to a 1.5-ml microcentrifuge tube.
2.
Add 30 µl of IVT Binding Buffer and vortex for 5 seconds.
3.
Add 130 µl of isopropanol and vortex for 5 seconds.
4.
Place an IVT RNA Clean-Up Spin Column in a Collection Tube and load the sample from Step 3
onto the column. Centrifuge at 11,000
g
for 30 seconds at room temperature.
5.
Discard the flowthrough from the Collection Tube and place the column back in the same Collection
Tube.
6.
Add 600 µl of IVT Wash Buffer and centrifuge at 11,000
g
for 30 seconds at room temperature.
7.
Discard the flowthrough from the Collection Tube and place the column back in the same Collection
Tube.
8.
Add 250 µl of IVT Wash Buffer and centrifuge at 11,000
g
for 2 min at room temperature.
9.
Place the IVT RNA Clean-Up Spin Column in a new 1.5-ml microcentrifuge tube.
10.
Add 20 µl of RNase Free Water directly onto the silica membrane of the Spin Column and incubate
for 1 min at room temperature.
NOTE:
With an elution volume of 20 µl, a concentration of >0.5 µg/µl is expected. This is
appropriate for applications such as transfection, electroporation, and
in vitro
cleavage assays. If you
need a higher concentration of sgRNA, the elution volume can be reduced to 5 µl (see Table I).
Table I. Examples of sgRNA concentration and yield using different elution volumes.
Elution volume*
5 µl
10 µl
20 µl
Concentration
2.2 µg/µl
1.3 µg/µl
0.7 µg/µl
Yield
11.2 µg
12.8 µg
13.2 µg
*Different elution volumes were used for purification of sgRNA using the Guide-it IVT RNA Clean-Up Kit. A
smaller elution volume can be used without a significant loss of total yield of sgRNA if the higher sgRNA
concentration is required for your experiment.
11.
Centrifuge at 11,000
g
for 1 min at room temperature.
12.
Use 1 µl to measure the OD using a NanoDrop Spectrophotometer (or equivalent). A yield of 10–20
µg of sgRNA is expected.
13.
Store sgRNA at –80°C until it is used to form the rCas9/sgRNA RNP complex.