RAPID Lite

CR2

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I. DNA amplification

Kit
GE:illustra GenomiPhi V2 DNA Amplification Kit

II. PCR

Primer details

Exon No.	Forward sequence	Reverse sequence	Expected PCR product size (bp)	Genomic regions amplified by ePCR with the primer
1	TAGCATAACACAGCATGGCAC	ACACGCGTCGAGCACTG	595bp	chr1:207627345+207627939
2	GAGTTTGGGAGTAAGCAGGGA	ACTGTTTGTCCTTCAGGAGGGT	630bp	chr1:207639717+207640346
3-4	CAATGTTGCCCAGGGAAGCCA	TGCTGAAGAACAGCAGCCTC	682bp	chr1:207641771+207642452
5	TCTCTGGGCAGTCTGGGGTA	GATGCTTTGGTGAAGCACATATCCT	379bp	chr1:207642338+207642716
6	TATCAAGCAGCATCTGGGGCA	AAGCCCACAACCCCTTCTTC	557bp	chr1:207642964+207643520
7-8	TACAGCTGACGCCAGAGTGGA	CATGTGACAGGCAAGCACCT	596bp	chr1:207643973+207644568
9	GTTGCTATTGCTTCTTGGCCTGA	CTGTGCAACCATAACTATTGGCAGA	328bp	chr1:207644658+207644985
10	CCCATACCGTCCAGGAAACA	ATCTGTGCCCCTCTTGCCTC	625bp	chr1:207646006+207646630
11-12	TGGATCTGGGATCTATTCAGGGT	GCCTCTAAAGGATGCCACCA	627bp	chr1:207646699+207647325
13	TCAACTCCTCTCTGCCAAAGTTC	CCAATGAGGGAGACGCCTAA	302bp	chr1:207647479+207647780
14	TCCTAGGTCATGGGAACATAGCTG	TTTAGGCAGAATGTCAAGTGCAGA	742bp	chr1:207647907+207648648
15	GCTTGTTGCTTCTGGCCTTC	TCACCAGATAGACTACAGCCTGAGA	486bp	chr1:207649477+207649962
16	TCAGTGCAGCTTTCCTTACTGAGA	AACCCAGGACTCTCCACGCT	403bp	chr1:207651101+207651503
17	CCATGAAACGTGGGGCTACA	GACCAAATCAAGCCAAACACCAAC	368bp	chr1:207652528+207652895
18	TAGGCCCTGCAATCAGTCAG	TCTTAAGTCCTACAGAAAGGGCTTC	320bp	chr1:207653248+207653567
19	CCATGTGTTGGGAATTATGGAAAGCA	CACCGTGAATGCTACGCTTATTG	287bp	chr1:207658715+207659001

For Exon(s):2,5,6,9,10,11-12,13,14,15,16,17,18,19

Kit TaKaRa GC-buffer & LA-Taq^TM

2xGC Buffer 1	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 x30 ※annealing condition
72℃, 90 sec
72℃, 5 min
4℃, ∞

For Exon(s):1

Kit TaKaRa Multiplex PCR Assay Kit^TM

MultiplexPCR Mix 2	5 µL
5pmol/µL Fw primer	1 µL
5pmol/µL Rev primer	1 µL
MultiplexPCR Mix 1	0.05 µL
Genomiphi product	0.5 µL
Sterilized distilled water	2.45 µL

↓
94℃, 30 sec
65℃, 1 min x30 ※annealing condition
4℃, ∞

For Exon(s):7-8

Kit TaKaRa GC-buffer & LA-Taq^TM

2xGC 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 x30 ※annealing condition
72℃, 90 sec
72℃, 5 min
4℃, ∞

For Exon(s):3-4

Kit TaKaRa PrimeSTAR GXL DNA Polymerase^TM

5xPrimeSTAR 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 x30 ※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 ÂµL is added directly to the PCR product 9 µL.
37℃, 30min
80℃, 15min

III. Sequencing reaction

Kit
BigDye® Terminator v3.1 Cycle Sequencing Kit
Primer details

Exon No.	Forward sequence	Reverse sequence	Expected PCR product size (bp)	Genomic regions amplified by ePCR with the primer
1	TAGCATAACACAGCATGGCAC	ACACGCGTCGAGCACTG	595bp	chr1:207627345+207627939
2	GAGTTTGGGAGTAAGCAGGGA	GTACAAAACGTGGTCTGCACA	610bp	chr1:207639717+207640326
3-4	CAATGTTGCCCAGGGAAGCCA	TGCTGAAGAACAGCAGCCTC	682bp	chr1:207641771+207642452
5	TCTCTGGGCAGTCTGGGGTA	GATGCTTTGGTGAAGCACATATCCT	379bp	chr1:207642338+207642716
6	TATCAAGCAGCATCTGGGGCA	AAGCCCACAACCCCTTCTTC	557bp	chr1:207642964+207643520
7-8	TACAGCTGACGCCAGAGTGGA	CATGTGACAGGCAAGCACCT	596bp	chr1:207643973+207644568
9	GTTGCTATTGCTTCTTGGCCTGA	CTGTGCAACCATAACTATTGGCAGA	328bp	chr1:207644658+207644985
10	CCCATACCGTCCAGGAAACA	ATCTGTGCCCCTCTTGCCTC	625bp	chr1:207646006+207646630
11-12	TGGATCTGGGATCTATTCAGGGT	GCCTCTAAAGGATGCCACCA	627bp	chr1:207646699+207647325
13	TCAACTCCTCTCTGCCAAAGTTC	CCAATGAGGGAGACGCCTAA	302bp	chr1:207647479+207647780
14	TCCTAGGTCATGGGAACATAGCTG	TTTAGGCAGAATGTCAAGTGCAGA	742bp	chr1:207647907+207648648
15	GCTTGTTGCTTCTGGCCTTC	TCACCAGATAGACTACAGCCTGAGA	486bp	chr1:207649477+207649962
16	TCAGTGCAGCTTTCCTTACTGAGA	AACCCAGGACTCTCCACGCT	403bp	chr1:207651101+207651503
17	CCATGAAACGTGGGGCTACA	GACCAAATCAAGCCAAACACCAAC	368bp	chr1:207652528+207652895
18	TAGGCCCTGCAATCAGTCAG	TCTTAAGTCCTACAGAAAGGGCTTC	320bp	chr1:207653248+207653567
19	CCATGTGTTGGGAATTATGGAAAGCA	CACCGTGAATGCTACGCTTATTG	287bp	chr1:207658715+207659001

IV. Ethanol precipitation

V. Sequencing analysis