Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
(030619)
takarabio.com
Takara Bio USA, Inc.
Page 13 of 24
Reagent
Amount (µl)
sgRNA PCR template (from Step B)
5
Guide-it In Vitro Transcription Buffer
7
Guide-it T7 Polymerase Mix
3
RNase Free Water
5
Total
20
NOTE:
If you require a higher amount of sgRNA, you can scale up the total reaction size (e.g., to
50 µl) without affecting the quality of the sgRNA.
2.
Place reactions in a preheated thermal cycler with a heated lid and run the following program:
37°C
4 hr
4°C
forever
NOTE:
We recommend a 4-hr incubation, but a shorter incubation time is acceptable if you do not
need to maximize your sgRNA yield (see Figure 7). We have observed a clear, sharp band via Agilent
Bioanalyzer following an 8-hr incubation, indicating no drop in sgRNA quality.
Figure 7. sgRNA yield over time.
20 µl of sgRNA IVT reactions were incubated for different times at 37°C. The reactions
were purified using the Guide-it IVT RNA Clean-Up Kit and quantified using a NanoDrop spectrophotometer.
3.
Following incubation, add 2 µl of Recombinant DNase I (RNase-Free) to the 20-µl IVT reaction.
Briefly vortex and spin down to collect the reagents at the bottom of the tube.
4.
Place reactions in a preheated thermal cycler with a heated lid and run the following program:
37°C
15 min
4°C
forever