Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
(030619)
takarabio.com
Takara Bio USA, Inc.
Page 12 of 24
PCR-Amplifying the sgRNA Template
For use with the Guide-it sgRNA In Vitro Transcription Components v2.
1.
Combine the following components in a 200-µl PCR tube. Briefly vortex and spin down to collect the
reagents at the bottom of the tube.
Reagent
Amount (
µl)
PrimeSTAR Max Premix (2X)
12.5
Guide-it Scaffold Template
1
Your forward primer (10 µM)
0.5
RNase Free Water
11
Total
25
2.
Place reactions in a preheated thermal cycler with a heated lid and run the following program:
33 cycles:
98°C
10 sec
68°C
10 sec
4°C
forever
3.
Run and analyze 5 µl of the PCR product on a 2% agarose gel with a 100-bp DNA ladder. You
should see a single band at ~130 bp (see Figure 6).
Figure 6. Gel electrophoresis of the PCR product.
M = 100-bp DNA ladder. S = 5 µl of sample.
C.
Performing the In Vitro Transcription (IVT) Reaction
The PCR product amplified in the previous section is directly used as template for the IVT reaction
without purification.
1.
Combine the following components in a 200-µl PCR tube. Briefly vortex and spin down to collect the
reagents at the bottom of the tube.
