2. Mix by vortexing for 15 s and incubate in a heat block at 65
C
for 6 min.
3. Mix by vortexing for 15 s and incubate in a heat block at 98
C
for 2 min.
4. PCR amplify the genomic region spanning the Cas9 target site
(HbE point mutation) using the primers described in Table 3
(and Fig. 3) using the PCR reaction in Table 7.
5. Transfer the PCR tubes to a thermal cycler using conditions
listed in Table 8.
6. After PCR, run the samples on 1% (w/v) agarose gel at 100 V
for 25 min.
7. Select the iPSC colonies with HbE negative based on the PCR
result (Fig. 4).
HbE mutant allele
Wild-type allele
A
G
HbE-Fc
HbE-Fm
HbE-Rc
HbE-Rn
Fig. 3 Primer design for multiplex PCR analysis
Table 7
Reaction setup for multiplex PCR analysis of hemoglobin E
Component
Amount (μL)
Final concentration
HotStarTaq polymerase, 5 units/μL
0.3
1.5 U
PCR buffer, 10Â
3
1 Â
MgCl 2 , 25 mM
1.8
1.5 mM
dNTPs, 2 mM
1.5
0.1 mM
HbE-Fc primer, 10 μM
1.2
0.4 μM
HbE-Rc primer, 10 μM
1.2
0.4 μM
HbE-Rn primer, 10 μM
1.5
0.5 μM
HbE-Fm primer, 10 μM
1.5
0.5 μM
DNA sample
2
Nuclease-free water
To 30
206
Methichit Wattanapanitch