Reaction

↓ Sample Genome 1 µL＋Sample Buffer 9 µL
↓ 95℃, 3min
↓ 4℃ or on ice
Making PreMix
↓ Reaction Buffer 9 µL＋Enzyme Mix 1 µL / Sample genome 1 µL
↓ Add PreMix 9 µL to each 1 sample (each tube)
↓ 30℃, 90 min
↓ 65℃, 10 min
↓ 4℃, ∞

For Exon(s): 2-3,4-5,7-8,9,10,11,12,13,14,15,16-17,18-19,22,23,24

Kit

TaKaRa GC-buffer & LA-Taq^TM

Reaction

2×GC Buffer 2	5 µL
2.5mM dNTP	1.6 µL
5pmol/µL Fw primer	1 µL
5pmol/µL Rev primer	1 µL
LA Taq	0.1 µL
Genomiphi product	0.5 µL
Sterilized distilled water	0.8 µL

↓
94℃, 1 min
94℃, 30 sec
57℃, 20 sec ×30 ※annealing condition
72℃, 90 sec
72℃, 5 min
4℃, ∞

For Exon(s): 6,20-21

Kit

TaKaRa PrimeSTAR GXL DNA Polymerase^TM

Reaction

5×PrimeSTAR GXL Buffer	2 µL
2.5mM dNTP	0.8 µL
5pmol/µL Fw primer	1 µL
5pmol/µL Rev primer	1 µL
PrimeSTAR GXL DNA Polymerase	0.2 µL
Genomiphi product	0.5 µL
Sterilized distilled water	4.5 µL

↓
94℃, 1 min
94℃, 30 sec
60℃, 20 sec ×30 ※annealing condition
72℃, 90 sec
72℃, 5 min
4℃, ∞

For Exon(s): 6,20-21

Kit

TaKaRa PrimeSTAR GXL DNA Polymerase^TM

Reaction

5×PrimeSTAR GXL Buffer	2 µL
2.5mM dNTP	0.8 µL
5pmol/µL Fw primer	1 µL
5pmol/µL Rev primer	1 µL
PrimeSTAR GXL DNA Polymerase	0.2 µL
Genomiphi product	0.5 µL
Sterilized distilled water	4.5 µL

↓
98℃, 10 sec
60℃, 15 sec
68℃, 1 min ×30 ※annealing condition
4℃, ∞

PCR Clean-Up Kit

ExoSAP-IT®

At first, ExoSAP-IT is diluted by 5% using sterilized distilled water (ExoSAP-IT 0.2 µL + Sterilized distilled water 3.8 µL / 1 sample ).
↓ The diluted ExoSAP-IT 4 µL is added directly to the PCR product 9 µL.

↓37℃, 30min
↓80℃, 15min

Reaction

2.5×Premix	0.5 µL
5×Sequence buffer	1.75 µL
5pmol/µL primer	0.5 µL
PCR product	0.5 µL
Sterilized distilled water	6.75 µL

↓
96℃, 1 min
96℃, 5 sec
50℃, 5 sec ×25
60℃, 4 min
4℃, ∞

↓ add 125mM EDTA(pH7.5-8.0) 1 µL, 3M sodium acetate 1 µL
↓ add 100% ethanol (-20℃) 25 µL
↓ vortex 2 min
↓ incubate at room temperature for 15 min
↓ centrifuge at 4000rpm for 30 min ( 4℃)
↓ centrifuge at 1000rpm for 1 sec (4℃) upside down
↓ The pellet is washed in cold 70% ethanol 35 µL.
↓ centrifuge at 4000rpm for 10 min (4℃)
↓ centrifuge at 1000rpm for 1 sec (4℃) upside down
↓3min dry up