Team:Kyoto/Notebook

• 
• Project
  • Description
  • Design
  　　　　
  • Results
  　　　　　　　　　
  • Demonstration
  • Discussion
• Modeling
• Lab Work
  • Notebook
  • Materials&Methods
  • Special Methods
  • Safety
• Parts
  • Basic Parts
  • Composite Parts
  • Improving parts
  • Parts Collection
• Human Practices
  • Human Practice/Silver
  • Integrated Human Practice/Gold
  • Collaborations
• Team
  • Members
  • Attributions

Notebook

July
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

August
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

September
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

October
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

07/10

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	shortAK2	shortAK2	B.xylophilus cDNA
2	Top1	Top1	B.xylophilus cDNA
3	long AK2	long AK2	B.xylophilus cDNA
4	eef-1g	eef-1g	B.xylophilus cDNA
5	AK1	AK1	B.xylophilus cDNA
6	Asb	Asb	B.xylophilus cDNA
7	14-3-3zeta	14-3-3zeta	B.xylophilus cDNA
8	tropomiosin	tropomiosin	B.xylophilus cDNA
9	14-3-3 protein	14-3-3 protein	B.xylophilus cDNA

PCR Polymerase : 5*Q5 Polymerase
98.0°C : 30sec
98.0°C : 10sec
58.0°C : 30sec
72.0°C : 1min
72.0°C : 1min
4.0°C : Infinate
X35 cycle
PCR pulification: lane 2~9
Gel extraction: lane 2~9

07/11

Miniprep_Nanodrop

Yoshimoto

sample	Sample name	260/280	260/230	ng/ul
1	2	no data	no data	237.8
2	3	no data	no data	180.5
3	4	no data	no data	185.6
4	5	no data	no data	167.2
5	6	no data	no data	328.2
6	7	no data	no data	133.7
7	8	no data	no data	168.4
8	9	no data	no data	242.5

The way of extraction : Gel extraction

07/11

Ligation

Yoshimoto

sample	Vector name	Vector amount(ul)	Insert name	Insert amount(ul)
1	YEplac195/sma1	1	Template2	1.0
2	YEplac195/sma1	1	Template3	1.0
3	YEplac195/sma1	1	Template4	1.0
4	YEplac195/sma1	1	Template5	1.0
5	YEplac195/sma1	1	Template6	1.0
6	YEplac195/sma1	1	Template7	1.0
7	YEplac195/sma1	1	Template8	1.0
8	YEplac195/sma1	1	Template9	1.0

07/11

Transformation

Yoshimoto

sample	CompetentCell name	DNA name
1	DH5a	7/11 ligation product

07/12

Ligation

Yoshimoto

sample	Vector name	Vector amount(ul)	Insert name	Insert amount(ul)
1	pRS315	1	Template	1.0

07/12

Transformation

Yoshimoto

sample	CompetentCell name	DNA name
1	DH5a	7/12 ligation product 2
2	DH5a	7/12 ligation product 3
3	DH5a	7/12 ligation product 4
4	DH5a	7/12 ligation product 5
5	DH5a	7/12 ligation product 6
6	DH5a	7/12 ligation product 7
7	DH5a	7/12 ligation product 8
8	DH5a	7/12 ligation product 9

07/13

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	T7Top1	T7Top1	7/10 gel extraction product2
2	T7long AK2	T7long AK2	7/10 gel extraction product3
3	T7eef-1g	T7eef-1g	7/10 gel extraction product4
4	T7AK1	T7AK1	7/10 gel extraction product5
5	T7Asb	T7Asb	7/10 gel extraction product6
6	T714-3-3zeta	T714-3-3zeta	7/10 gel extraction product7
7	T7tropomiosin	T7tropomiosin	7/10 gel extraction product8
8	T714-3-3 protein	T714-3-3 protein	7/10 gel extraction product9

PCR Polymerase : ExTaq
98.0°C : 1min
98.0°C : 10sec
58.0°C : 30sec
72.0°C : 1min
72.0°C : 1min
4.0°C : Infinate
X35 cycle

07/14

Miniprep_Nanodrop

Yoshimoto

sample	Sample name	260/280	260/230	ng/ul
1	1	1.84	1.96	98.4
2	2	1.79	1.85	76.2
3	3	1.85	2.1	102.1
4	4	1.62	0.49	9.5
5	5	1.91	1.91	83.6
6	6	1.82	1.73	48.0
7	7	1.85	1.85	83.0
8	8	1.86	2.84	81.5
9	9	1.57	0.33	22.6
10	10	1.54	0.55	17.8
11	11	1.83	2.02	88.0
12	12.0	1.85	2.94	82.0
13	13.0	1.83	1.83	90.6
14	14.0	1.87	1.65	46.2
15	15.0	1.89	2.0	75.8
16	16.0	1.67	1.98	114.0
17	17.0	1.88	1.46	88.5
18	18.0	1.85	1.91	99.4
19	19.0	1.79	1.79	53.8
20	20.0	1.37	0.58	17.0
21	21.0	1.63	0.57	0.57
22	22.0	1.83	1.84	53.0

The way of extraction : promega

07/15

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	1	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
2	2	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
3	3	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
4	4	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
5	5	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
6	6	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
7	7	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
8	8	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
9	9	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
10	10	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
11	11	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
12	12	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
13	13	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
14	14	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
15	15	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
16	16	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
17	17	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
18	18	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
19	19	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
20	20	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
21	21	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0
22	22	5.0	Pst1	0.05	Sac1	0.05	10xM	1.0	10.0

07/16

Miniprep_Nanodrop

Yoshimoto

sample	Sample name	260/280	260/230	ng/ul
1	T7 3	1.86	2.28	255.7
2	T7 4	1.83	2.13	121.9
3	T7 5	1.83	2.29	224.2
4	T7 6	1.85	2.18	146.1
5	T7 7	1.85	2.25	96.5
6	T7 8	1.85	2.27	165.4
7	T7 9	No data	No data	No data

The way of extraction : promega

07/16

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	short AK2	short AK2	B.xylophilus cDNA
2	Top1	Top1	B.xylophilus cDNA

PCR Polymerase : Extaq
94.0°C : 1min
94.0°C : 30sec
60.0°C : 1min
72.0°C : 2min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/06

Nebuilder

Yoshimoto

Sample	Vectorname	Vector amount (ul)	Insert1 name	Insert1 amount(ul)	Insert2 name	Insert2 amount (ul)	Mastermix amount (ul)	Total amount(ul)
1	YEplac195/sma1	2.5	Gal-AK	0.8	Ter-AK	1.0	10.0	20.0
2	YEplac195/sma1	2.5	GPD-AK	1.2	Ter-AK	1.0	10.0	20.0

08/06

Transformation

Yoshimoto

sample	CompetentCell name	DNA name
1	DH5a	Insert

08/07

Miniprep_Nanodrop

Yoshimoto

sample	Sample name	260/280	260/230	ng/ul
1	1	1.97	2.03	36.0
2	2	2.03	1.89	28.7
3	3	1.92	1.98	34.3
4	4	1.96	1.88	35.5
5	5	2.03	1.87	16.2
6	6	2.01	2.11	50.0
7	7	1.95	1.95	30.3
8	8	1.95	2.06	38.2
9	9	2.12	1.94	17.0
10	10	no data	no data	no data
11	11	1.99	1.92	27.9
12	12	1.96	1.79	19.1
13	13	1.95	1.96	29.4
14	14	1.98	1.64	22.7

The way of extraction : fastgene plasmid mini kit

08/08

Transformation

Yoshimoto

sample	CompetentCell name	DNA name
1	DH5a	8/6 Nebuilder product
2	DH5a	8/6 Nebuilder product
3	DH5a	8/6 Nebuilder product
4	DH5a	8/6 Nebuilder product
5	DH5a	8/6 Nebuilder product
6	DH5a	8/6 Nebuilder product
7	DH5a	8/6 Nebuilder product
8	DH5a	8/6 Nebuilder product

08/08

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	miniprep 1	5.0	Pst1	0.05	10xH	1.0	10.0
2	miniprep 2	5.0	Pst1	0.05	10xH	1.0	10.0
3	miniprep 3	5.0	Pst1	0.05	10xH	1.0	10.0
4	miniprep 4	5.0	Pst1	0.05	10xH	1.0	10.0
5	miniprep 5	5.0	Pst1	0.05	10xH	1.0	10.0
6	miniprep 7	5.0	Pst1	0.05	10xH	1.0	10.0

08/09

Miniprep_Nanodrop

Yoshimoto

sample	Sample name	260/280	260/230	ng/ul
1	8	1.69	1.32	12.1
2	9	1.97	1.45	17.0
3	11	1.83	0.99	12.5
4	12	1.92	1.05	12.3

The way of extraction : promega PureYield Plasmid Midiprepkit

08/09

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	8/8 miniprep8	5.0	Pst1	0.05	10xH	1.0	10.0
2	8/8 miniprep9	5.0	Pst1	0.05	10xH	1.0	10.0
3	8/8 miniprep11	5.0	Pst1	0.05	10xH	1.0	10.0
4	8/8 miniprep12	5.0	Pst1	0.05	10xH	1.0	10.0

08/09

Restriction Enzyme digestion

Fukuda

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	AK2	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
2	AK2	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
3	eef-1g	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
4	eef-1g	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
5	AK1	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
6	AK1	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
7	Asb	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
8	Asb	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
9	14-3-3-zeta	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
10	14-3-3-zeta	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
11	tropomiosin	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
12	tropomiosin	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
13	14-3-3 protein	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0
14	14-3-3 protein	5.0	Xba1	0.5	EcoR1	0.5	10xM	6.0	36.0

08/10

Nebuilder

Yoshimoto

Sample	Vectorname	Vector amount (ul)	Insert1 name	Insert1 amount(ul)	Insert2 name	Insert2 amount (ul)	Mastermix amount (ul)	Total amount(ul)
1	YEplac195/sma1	2.5	Gal-AK	0.8	Ter-AK	1.0	10.0	20.0
2	YEplac195/sma1	2.5	GPD-AK	1.2	Ter-AK	1.0	10.0	20.0

08/10

Transformation

Yoshimoto

sample	CompetentCell name	DNA name
1	DH5a	insert

08/10

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	T7AK2F	T7AK2R	B.xylophils cDNA1
2	T7AK2F	T7AK2R	B.xylophils cDNA2
3	T7Egf-1gF	T7Egf-1gR	B.xylophils cDNA3
4	T7Egf-1gF	T7Egf-1gR	B.xylophils cDNA4
5	T7AK1F	T7Ak1R	B.xylophils cDNA5
6	T7AK1F	T7Ak1R	B.xylophils cDNA6
7	T714-3-3zetaF	T714-3-3 zetaR	B.xylophils cDNA7
8	T714-3-3zetaF	T714-3-3 zetaR	B.xylophils cDNA8
9	T7tropomiosin F	T7tropomiosin R	B.xylophils cDNA9
10	T7tropomiosin F	T7tropomiosin R	B.xylophils cDNA10
11	T714-3-3 F	T714-3-3 R	B.xylophils cDNA11
12	T714-3-3 F	T714-3-3 R	B.xylophils cDNA12
13	T7AK2F	T7AK2R	water

PCR Polymerase : gotaq
94.0°C : 2min
94.0°C : 30sec
62.0°C : 30sec
68.0°C : 1min20sec
68.0°C : 7min
4.0°C : Infinate
X30 cycle

08/11

colony_PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	1
2	M13F	M13R	3
3	M13F	M13R	5
4	M13F	M13R	7
5	M13F	M13R	9
6	M13F	M13R	11
7	M13F	M13R	13
8	M13F	M13R	15
9	M13F	M13R	17
10	M13F	M13R	19
11	M13F	M13R	21
12	M13F	M13R	23
13	M13F	M13R	25
14	M13F	M13R	27
15	M13F	M13R	29
16	M13F	M13R	31
17	M13F	M13R	33
18	M13F	M13R	35
19	M13F	M13R	37
20	M13F	M13R	39
21	M13F	M13R	41
22	M13F	M13R	43
23	M13F	M13R	45
24	M13F	M13R	47
25	M13F	M13R	49

PCR Polymerase : QuickTaq
94.0°C : 2min
94.0°C : 30sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 1min
4.0°C : Infinate
X35 cycle

08/11

Restriction Enzyme digestion

Ohara

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	1-1	5.0	Pst1	1.0	10xH	1.0	3.0
2	1-2	5.0	Pst1	1.0	10xH	1.0	3.0
3	1-3	5.0	Pst1	1.0	10xH	1.0	3.0
4	1-4	5.0	Pst1	1.0	10xH	1.0	3.0
5	1-5	5.0	Pst1	1.0	10xH	1.0	3.0
6	2-1	5.0	Pst1	1.0	10xH	1.0	3.0
7	1-6	5.0	Pst1	1.0	10xH	1.0	3.0
8	2-2	5.0	Pst1	1.0	10xH	1.0	3.0
9	2-3	5.0	Pst1	1.0	10xH	1.0	3.0
10	2-4	5.0	Pst1	1.0	10xH	1.0	3.0
11	2-5	5.0	Pst1	1.0	10xH	1.0	3.0
12	2-6	5.0	Pst1	1.0	10xH	1.0	3.0
13	3-1	5.0	Pst1	1.0	10xH	1.0	3.0
14	3-2	5.0	Pst1	1.0	10xH	1.0	3.0
15	3-3	5.0	Pst1	1.0	10xH	1.0	3.0
16	3-4	5.0	Pst1	1.0	10xH	1.0	3.0

08/11

Miniprep_Nanodrop

Yoshimoto

sample	Sample name	260/280	260/230	ng/ul
1	1	1.88	1.6	116.8
2	8	1.9	1.67	170.0
3	9	1.91	1.71	167.6
4	12	1.88	1.6	135.6

The way of extraction : promega PureYield Plasmid Midiprepkit

08/11

colony_PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	2
2	M13F	M13R	4
3	M13F	M13R	6
4	M13F	M13R	8
5	M13F	M13R	10
6	M13F	M13R	12
7	M13F	M13R	14
8	M13F	M13R	16
9	M13F	M13R	18
10	M13F	M13R	20
11	M13F	M13R	22
12	M13F	M13R	24
13	M13F	M13R	26
14	M13F	M13R	28
15	M13F	M13R	30
16	M13F	M13R	32
17	M13F	M13R	34
18	M13F	M13R	36
19	M13F	M13R	38
20	M13F	M13R	40
21	M13F	M13R	42
22	M13F	M13R	44
23	M13F	M13R	46
24	M13F	M13R	48
25	M13F	M13R	50
26	M13F	M13R	53
27	M13F	M13R	54
28	M13F	M13R	55
29	M13F	M13R	56

PCR Polymerase : Quick_taq
94.0°C : 2min
94.0°C : 30sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 10min
4.0°C : Infinate
X35 cycle

08/11

Ligation

Yoshimoto

sample	Vector name	Vector amount(ul)	Insert name	Insert amount(ul)
1	YEplac195/sma1	1	AK2-gal1	1.0
2	YEplac195/sma1	1	AK2-GPD	1.0

08/11

Restriction Enzyme digestion

Ohara

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	7A	5.0	Pst1	0.5	10xH	6.0	60.0
2	7B	5.0	Pst1	0.5	10xH	6.0	60.0

08/11

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	AK2-gal1 Nebuilder
2	M13F	M13R	AK2 GPD Nebuilder
3	M13F	M13R	gfp gal Nebuilder
4	M13F	M13R	gfp GPD Nebuilder
5	M13F	M13R	AK1 gal Nebuilder
6	M13F	M13R	AK1 GPD Nebuilder

PCR Polymerase : 2*KAPA 2G Robust HS Readymix
94.0°C : 2min
94.0°C : 15sec
60.0°C : 15sec
72.0°C : 40sec
72.0°C : 1min
4.0°C : Infinate
X35 cycle

08/11

Transformation

Yoshimoto

sample	CompetentCell name	DNA name
1	DH5a	insert

08/12

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	AK1F	AK1R	8
2	AK1F	AK1R	9
3	AK1F	AK1R	12
4	AK1F	AK1R	8/6 gal
5	AK1F	AK1R	8/6 GPD
6	AK1F	AK1R	gal1 AK1
7	AK1F	AK1R	gal1 AK1
8	AK1F	AK1R	water

PCR Polymerase : quicktaq
94.0°C : 2min
94.0°C : 30sec
60.0°C : 30sec
68.0°C : 1min
68.0°C : 7min
4.0°C : Infinate
X35 cycle

08/12

Ligation

Yoshimoto

sample	Vector name	Vector amount(ul)	Insert name	Insert amount(ul)
1	YEplac195/sma1	1	gblock 71	1.0
2	YEplac195/sma1	1	gblock 72	1.0
3	YEplac195/sma1	1	gblock 73	1.0
4	YEplac195/sma1	1	gblock 74	1.0
5	YEplac195/sma1	1	gblock 75	1.0
6	YEplac195/sma1	1	gblock 76	1.0
7	YEplac195/sma1	1	gblock 77	1.0
8	YEplac195/sma1	1	gblock 78	1.0

08/12

Transformation

Yoshimito

sample	CompetentCell name	DNA name
1	DH5a	insert

08/13

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	AK2F	AK2F	8
2	AK2F	AK2F	9
3	AK2F	AK2F	12
4	AK2F	AK2F	8/6 gal
5	AK2F	AK2F	8/6 GPD
6	AK2F	AK2F	7
7	AK2F	AK2F	water

PCR Polymerase : 2*KAPA hifi HS ready mix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 2min
72.0°C : 2min
4.0°C : Infinate
X40 cycle

08/13

Restriction Enzyme digestion

Ohara

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	1	5.0	Pst1	1.0	10xH	1.0	10.0
2	2	5.0	Pst1	1.0	10xH	1.0	10.0
3	3	5.0	Pst1	1.0	10xH	1.0	10.0
4	4	5.0	Pst1	1.0	10xH	1.0	10.0
5	5	5.0	Pst1	1.0	10xH	1.0	10.0
6	7	5.0	Pst1	1.0	10xH	1.0	10.0
7	1	5.0	Pst1	1.0	10xH	1.0	10.0
8	7	5.0	Pst1	1.0	10xH	1.0	10.0
9	9	5.0	Pst1	1.0	10xH	1.0	10.0
10	8	5.0	Pst1	1.0	10xH	1.0	10.0
11	12	5.0	Pst1	1.0	10xH	1.0	10.0

08/13

Restriction Enzyme digestion

Ohara

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	14	5.0	Pst1	1.0	10xH	1.0	10.0
2	16	5.0	Pst1	1.0	10xH	1.0	10.0
3	22	5.0	Pst1	1.0	10xH	1.0	10.0
4	24	5.0	Pst1	1.0	10xH	1.0	10.0
5	26	5.0	Pst1	1.0	10xH	1.0	10.0
6	30	5.0	Pst1	1.0	10xH	1.0	10.0
7	36	5.0	Pst1	1.0	10xH	1.0	10.0
8	24	5.0	Pst1	1.0	10xH	1.0	10.0
9	14	5.0	Pst1	0.0	10xH	0.0	10.0
10	22	5.0	Pst1	0.0	10xH	0.0	10.0
11	36	5.0	Pst1	0.0	10xH	0.0	10.0

08/13

colony PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	77-1
2	M13F	M13R	77-2
3	M13F	M13R	77-3
4	M13F	M13R	78-4
5	M13F	M13R	78-5

PCR Polymerase : 2*KAPA hifi HS ready mix
95.0°C : 1min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min
72.0°C : 1min
4.0°C : Infinate
X35 cycle

08/13

Miniprep_Nanodrop

Yoshimoto

sample	Sample name	260/280	260/230	ng/ul
1	14	1.91	1.44	82.6
2	16	1.96	1.67	91.4
3	22	2.04	1.77	96.7
4	24	1.88	1.5	93.0
5	26	1.89	1.51	89.4
6	30	1.9	1.48	74.6
7	36	1.91	1.5	87.5

The way of extraction : promega PureYield Plasmid Miniprepkit

08/13

Ligation

Yoshimoto

sample	Vector name	Vector amount(ul)	Insert name	Insert amount(ul)
1	YEplac195/sma1	1	Gal-AK2 99	2.0
2	YEplac195/sma1	1	GPD-AK2 80	2.0
3	YEplac195/sma1	1	Ter-AK2 81	2.0
4	YEplac195/sma1	1	T7 AK1 82	2.0
5	YEplac195/sma1	1	T7 AK1.2 83	2.0
6	YEplac195/sma1	1	Gal1 gfp 85	2.0
7	YEplac195/sma1	1	ter gfp 86	2.0

08/13

Nebuilder

Yoshimoto

Sample	Vectorname	Vector amount (ul)	Insert1 name	Insert1 amount(ul)	Insert2 name	Insert2 amount (ul)	Mastermix amount (ul)	Total amount(ul)
1	YEPlac195/sma1	0.25	GPD-AK	1.5	Ter-AK	5.0	5.0	12.0
2	YEPlac195/sma1	0.25	Gal-AK	2.0	Ter-AK	5.0	5.0	12.0

08/13

Transformation

Yoshimoto

sample	CompetentCell name	DNA name
1	DH5a	Nebuilder product
2	DH5a	Nebuilder product
3	DH5a	Nebuilder product
4	DH5a	Nebuilder product

08/14

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	79-1
2	M13F	M13R	80-1
3	M13F	M13R	80-2
4	M13F	M13R	81-1
5	M13F	M13R	97-1
6	M13F	M13R	98-1
7	M13F	M13R	98-2
8	M13F	M13R	98-3
9	M13F	M13R	92-1
10	M13F	M13R	99-1
11	M13F	M13R	100-1
12	M13F	M13R	101-1
13	M13F	M13R	101-2
14	M13F	M13R	101-3
15	M13F	M13R	102-1
16	M13F	M13R	water

PCR Polymerase : 2*KAPA hifi HS readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min20sec
72.0°C : 1min
4.0°C : Infinate
X35 cycle

08/14

Restriction Enzyme digestion

Ohara

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	2	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
2	3	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
3	4	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
4	5	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
5	7	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
6	14	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
7	16	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
8	22	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
9	77-1	5.0	Pst1	1.0	EcoR1	1.0	10xH	1.0	10.0
10	77-2	5.0	Pst1	1.0	EcoR1	1.0	10xH	1.0	10.0
11	77-3	5.0	Pst1	1.0	EcoR1	1.0	10xH	1.0	10.0
12	24	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
13	26	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
14	30	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
15	36	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
16	78-1	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
17	78-2	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
18	78	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
19	99	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
20	100	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
21	101	5.0	Pst1	1.0	None	None	10xH	1.0	10.0

08/14

colony_PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	97
2	M13F	M13R	98
3	M13F	M13R	99
4	M13F	M13R	100
5	M13F	M13R	101
6	M13F	M13R	102

PCR Polymerase : 2*KAPA hifi HS readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min20sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/14

Transformation

Yoshimoto

sample	CompetentCell name	DNA name
1	DH5a	Nebuilder product gal1-gfp
2	DH5a	Nebuilder product GPD-gfp
3	DH5a	8/13 ligation product Gal-AK2
4	DH5a	8/13 ligation product GPD-AK2
5	DH5a	8/13 ligation product Ter-AK2

08/14

Transformation

Yoshimoto

sample	CompetentCell name	DNA name
1	DH5a	ligation product Gal-AK2
2	DH5a	ligation product GPD-AK2
3	DH5a	ligation product Ter-AK2

08/14

Miniprep_Nanodrop

Yoshimoto

sample	Sample name	260/280	260/230	ng/ul
1	101	1.97	0.93	24.9
2	100	1.99	0.99	34.8
3	97	1.94	1.35	50.1
4	98	1.97	1.97	40.3
5	99.0	1.96	1.65	99.7
6	73.5	1.82	0.9	47.9
7	78.4	1.86	0.99	43.7
8	71-3	1.88	1.21	67.4
9	36	1.88	1.41	129.5
10	77-1	1.81	0.99	70.8
11	77-2	1.89	1.2	63.0
12	30	1.82	1.23	135.8
13	26	1.86	1.31	143.9
14	24	1.88	1.43	141.5
15	14	1.82	1.31	178.7
16	16	1.91	1.37	132.3
17	22	1.79	1.35	207.8
18	7	1.85	1.28	94.4
19	5	1.79	0.91	63.8
20	4	1.66	0.81	64.2
21	2	1.87	0.96	50.5
22	3	1.79	1.15	67.1

The way of extraction : promega

08/15

Restriction Enzyme digestion

Ohara

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	92	5.0	Pst1	1.0	10xH	1.0	10.0
2	97	5.0	Pst1	1.0	10xH	1.0	10.0
3	98	5.0	Pst1	1.0	10xH	1.0	10.0
4	100	5.0	Pst1	1.0	10xH	1.0	10.0
5	101	5.0	Pst1	1.0	10xH	1.0	10.0
6	92	5.0	Pst1	1.0	10xH	1.0	10.0
7	97	5.0	Pst1	1.0	10xH	1.0	10.0
8	98	5.0	Pst1	1.0	10xH	1.0	10.0
9	100	5.0	Pst1	1.0	10xH	1.0	10.0
10	101	5.0	Pst1	1.0	10xH	1.0	10.0

08/15

Nebuilder

Yoshimoto

Sample	Vectorname	Vector amount (ul)	Insert1 name	Insert1 amount(ul)	Insert2 name	Insert2 amount (ul)	Mastermix amount (ul)	Total amount(ul)
1	YEplac195/sma1	0.25	Gal-AK	1.825	Ter-AK	1.5	5.0	10.0
2	YEplac195/sma1	0.25	GPD-AK	1.25	Ter-AK	1.5	5.0	10.0

08/15

Transformation

Yoshimoto

sample	CompetentCell name	DNA name
1	DH5a	111
2	DH5a	112

08/17

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK25	IK26	Rev
2	IK27	IK22	yeast genome
3	IK23	IK28	yeast genome

PCR Polymerase : 2*KAPA hifi HS Ready mix
98.0°C : 3min
98.0°C : 20sec
62.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/17

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK27	IK22	yeast genome
2	IK23	IK28	yeast genome
3	IK27	IK28	GPDp+Rev+GPDt

PCR Polymerase : 2*KAPA hifi HS ready mix
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/17

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK25	IK26	Rev
2	IK27	IK22	yeast genome
3	IK23	IK28	yeast genome
4	IK25	IK26	Rev after purification
5	IK27	IK22	GPDp after purification
6	IK23	IK28	GPDt after purification

PCR Polymerase : 2*KAPA hifi HS ready mix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X36 cycle

08/17

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	86-1
2	M13F	M13R	86-2
3	M13F	M13R	81-1
4	M13F	M13R	81-2
5	M13F	M13R	81-3
6	M13F	M13R	95-1
7	M13F	M13R	95-2
8	M13F	M13R	95-3
9	M13F	M13R	95-4
10	M13F	M13R	93-1
11	M13F	M13R	93-2
12	M13F	M13R	92
13	M13F	M13R	brue colony
14	M13F	M13R	11

PCR Polymerase : 2*KAPA hifi HS readymix
95.0°C : 3min
95.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min20sec
72.0°C : 1min
4.0°C : Infinate
X35 cycle

08/17

Nebuilder

Yoshimoto

Sample	Vectorname	Vector amount (ul)	Insert1 name	Insert1 amount(ul)	Insert2 name	Insert2 amount (ul)	Mastermix amount (ul)	Total amount(ul)
1	YEplac195/sma1	0.8	Gal-AK2	6.5	Ter-AK2	5.0	10.0	22.3
2	YEplac195/sma1	0.8	GPD-AK2	4.2	Ter-AK2	5.0	10.0	20.0

08/17

Transformation

Yoshimoto

sample	CompetentCell name	DNA name
1	BL21	113
2	BL21	114
3	BL21	115

08/18

Restriction Enzyme digestion

Ohara

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	79-2	5.0	Pst1	1.0	EcoR1	1.0	10xH	1.0	10.0
2	79-3	5.0	Pst1	1.0	EcoR1	1.0	10xH	1.0	10.0
3	79-6	5.0	Pst1	1.0	EcoR1	1.0	10xH	1.0	10.0
4	80-2	5.0	Pst1	1.0	EcoR1	1.0	10xH	1.0	10.0
5	80-7	5.0	Pst1	1.0	EcoR1	1.0	10xH	1.0	10.0
6	80-13	5.0	Pst1	1.0	EcoR1	1.0	10xH	1.0	10.0
7	81-1	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
8	81-2	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
9	97	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
10	99-A	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
11	99-B	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
12	100	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
13	100-1	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
14	100-2	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
15	101-1	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
16	101 3	5.0	Pst1	1.0	None	None	10xH	1.0	10.0
17	102	5.0	Pst1	1.0	None	None	10xH	1.0	10.0

08/18

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK27	IK28	Rev

PCR Polymerase : 2*KAPA hifi HS Ready mix
98.0°C : 3min
98.0°C : 20sec
60.0°C : 20sec
72.0°C : 1min20sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/18

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	M13F	AK2R	79-2
2	M13F	AK2R	79-3
3	M13F	AK2R	80-13
4	M13F	AK2F	79-2
5	M13F	AK2F	79-3
6	M13F	AK2F	80-13
7	M13R	M13F	blue colony

PCR Polymerase : 2*KAPA hifi HS readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/18

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK22	IK27	GPDp after purification
2	IK22	IK27	GPDp after purification
3	IK22	IK27	GPDp after purification
4	IK22	IK27	GPDp after purification
5	M13F	AK2R	113gal
6	M13F	AK2R	114GPD

PCR Polymerase : 2*KAPA hifi HS readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle
PCR pulification: lane1~4
Gel extraction: lane1~4

08/19

Ligation

Yoshimoto

sample	Vector name	Vector amount(ul)	Insert name	Insert amount(ul)
1	141	0.34	2T7akRRE3'	9.4
2	142	0.34	2T7ak1stem	8.3
3	143	0.34	1T7ak1stem	4.4
4	144	0.34	2T7RREak1	5.3
5	145	0.34	1T7RREak1	8.5
6	146	0.34	GPD+AK2	13.2
7	147	0.34	Gal1+AK2	15.8
8	148	0.34	AK2+ter	20.1

08/20

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK22	IK27	GPDp
2	IK22	IK27	GPDp
3	IK22	IK27	yeast genome
4	IK22	IK27	yeast genome
5	IK22	IK27	yeast genome
6	IK22	IK27	yeast genome
7	IK27	IK28	GPDp+Rev+GPDt
8	IK27	IK28	GPDp+Rev+GPDt

PCR Polymerase : 2*KAPA hifi HS ready mix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/20

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK27	IK22	GPDp
2	IK21	IK22	GPDp

PCR Polymerase : 2*KAPA HS readymix
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/21

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK29	IK30	Rev
2	IK29	IK30	Rev
3	IK29	IK30	Rev
4	IK29	IK30	Rev
5	M13R	AK1R	141 1-1
6	M13R	AK1R	141 1-2
7	M13R	AK1R	141 1-3
8	M13R	AK1R	141 1-4
9	M13R	AK1R	142 1-1
10	M13R	AK1R	142 1-2
11	M13R	AK1R	142 1-3
12	M13R	AK1R	142 1-4
13	M13R	AK1F	141 1-1
14	M13R	AK1F	141 1-2
15	M13R	AK1F	141 1-3
16	M13R	AK1F	141 1-4
17	M13R	AK1F	142 1-1
18	M13R	AK1F	142 1-2
19	M13R	AK1F	142 1-3
20	M13R	AK1F	142 1-4

PCR Polymerase : 2*KAPA hifi HS Readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min20sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/21

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	M13F	AK1R	141 2-1
2	M13F	AK1R	141 2-4
3	M13F	AK1R	141 1-15
4	M13F	AK1R	142 1-5
5	M13F	AK1R	143 9
6	M13F	AK1R	143 13
7	M13F	AK1R	143 27
8	M13F	AK1R	143 2
9	M13F	AK1F	141 2-1
10	M13F	AK1F	141 2-4
11	M13F	AK1F	141 1-15
12	M13F	AK1F	142 1-5
13	M13F	AK1F	143 9
14	M13F	AK1F	143 13
15	M13F	AK1F	143 27
16	M13F	AK1F	143 2

PCR Polymerase : 2*KAPA hifi HS Readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min20sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/21

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK29	IK30	Rev
2	IK29	IK30	Rev
3	IK29	IK30	Rev
4	IK29	IK30	Rev

PCR Polymerase : 2*KAPA hifi HS Readymix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min20sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle
PCR pulification: lane2~4
Gel extraction: lane2~4

08/22

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK21	IK22	GPDp
2	IK21	IK22	GPDp
3	IK21	IK22	GPDp
4	IK21	IK22	GPDp
5	IK21	IK22	water
6	IK23	IK28	GPDt
7	IK23	IK28	GPDt
8	IK23	IK28	GPDt
9	IK23	IK28	GPDt
10	IK23	IK28	water

PCR Polymerase : 2*KAPA hifi HS Readymix
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle
PCR pulification: lane2~4
Gel extraction: lane2~4

08/22

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK27	IK28	GPDp+Rev+GPDt
2	IK27	IK28	GPDp+Rev+GPDt
3	IK23	IK24	yeast genome
4	IK23	IK24	yeast genome
5	IK23	IK24	yeast genome
6	IK23	IK24	yeast genome
7	IK21	IK24	GPDp+Rev+GPDt

PCR Polymerase : 2*KAPA hifi HS ReadyMIx
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/22

PCR

Fukuda

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	144 11
2	M13F	M13R	144 3
3	M13F	M13R	144 7
4	M13F	M13R	144 1
5	M13F	M13R	144 17
6	M13F	M13R	144 5
7	M13F	M13R	144 9
8	M13F	M13R	144 13
9	M13F	M13R	142 1
10	M13F	M13R	142 5
11	M13F	M13R	142 3
12	M13F	M13R	142 4
13	M13F	M13R	142 2-1
14	M13F	M13R	142 2-2
15	M13F	M13R	142 2-3
16	M13F	M13R	142 2-4
17	M13F	M13R	143 13
18	M13F	M13R	143 15
19	M13F	M13R	143 5
20	M13F	M13R	153 21
21	M13F	M13R	143 17
22	M13F	M13R	143 19
23	M13F	M13R	143 25
24	M13F	M13R	143 9

PCR Polymerase : 2*KAPA hifi HS ReadyMix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/22

PCR

Takimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	145 1-13
2	M13F	M13R	145 2-13
3	M13F	M13R	145 2-15
4	M13F	M13R	145 1-5
5	M13F	M13R	145 2-17
6	M13F	M13R	145 2-11
7	M13F	M13R	145 2-9
8	M13F	M13R	145 1-17
9	M13F	M13R	146 2-3
10	M13F	M13R	146 2-1
11	M13F	M13R	146 1-3
12	M13F	M13R	146 2-7
13	M13F	M13R	146 2-9
14	M13F	M13R	146 2-5
15	M13F	M13R	146 1-4

PCR Polymerase : 2*KAPA hifi HS ReadyMix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/22

Nebuilder

Yoshimoto

Sample	Vectorname	Vector amount (ul)	Insert1 name	Insert1 amount(ul)	Insert2 name	Insert2 amount (ul)	Mastermix amount (ul)	Total amount(ul)
1	171.0	Vector amount (ul)	Gal1+gfp-2	0.78	Gal1+gfp	1.03	10.0	Total amount(ul)
2	172.0	Vector amount (ul)	Gal1+AK2	1.26	Gal1+AK2-2	1.01	10.0	Total amount(ul)
3	173.0	Vector amount (ul)	164.0	0.65	165.0	0.57	10.0	Total amount(ul)
4	174.0	Vector amount (ul)	161.0	0.65	162.0	0.76	10.0	Total amount(ul)
5	175.0	Vector amount (ul)	161.0	0.38	167.0	0.83	10.0	Total amount(ul)
6	176.0	Vector amount (ul)	166.0	0.38	165.0	0.83	10.0	Total amount(ul)
7	177.0	Vector amount (ul)	163.0	0.38	162.0	0.83	10.0	Total amount(ul)
8	178.0	Vector amount (ul)	163.0	0.38	167.0	0.83	10.0	Total amount(ul)

08/22

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	None	None	GPDp+Rev+GPDt
2	IK27	IK28	lane1 after 15 cycle

PCR Polymerase : 2*KAPA hifi Hs ReadyMix
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/22

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK23	IK28	GPDt
2	IK23	IK28	GPDt
3	IK23	IK28	GPDt
4	IK23	IK28	GPDt
5	IK27	IK30	GPDp + Rev
6	IK21	IK30	GPDp + Rev

PCR Polymerase : 2*KAPA hifi Robust
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/23

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	147 2-17
2	M13F	M13R	147 1-2
3	M13F	M13R	147 2-3
4	M13F	M13R	147 7
5	M13F	M13R	141 1-1
6	M13F	M13R	141 1-2
7	M13F	M13R	141 1-4
8	M13F	M13R	148 2-3
9	M13F	M13R	148 2-5
10	M13F	M13R	148 2-4
11	M13F	M13R	148 1-5
12	M13F	M13R	148 1-3
13	M13F	M13R	144 11
14	M13F	M13R	144 3
15	M13F	M13R	144 1
16	M13F	M13R	146 1-6
17	M13F	M13R	146 1-4
18	M13F	M13R	146 2-5
19	M13F	M13R	146 2-7
20	M13F	M13R	142 2-2
21	M13F	M13R	142 1-1
22	M13F	M13R	142 2-7
23	M13F	M13R	142 2-3
24	M13F	M13R	145 2-11
25	M13F	M13R	145 1-17
26	M13F	M13R	145 2-9
27	M13F	M13R	145 2-17
28	M13F	M13R	143 5
29	M13F	M13R	143 17
30	M13F	M13R	143 15
31	M13F	M13R	143 21
32	M13F	AK1R	147 2-17
33	M13F	AK1R	147 1-2
34	M13F	AK1R	147 2-3
35	M13F	AK1R	147 7
36	M13F	AK1R	141 1-1
37	M13F	AK1R	141 1-2
38	M13F	AK1R	141 1-4
39	M13F	AK1R	148 2-3
40	M13F	AK1R	148 2-5
41	M13F	AK1R	148 2-4
42	M13F	AK1R	148 1-5
43	M13F	AK1R	148 1-3
44	M13F	AK1R	144 11
45	M13F	AK1R	144 3
46	M13F	AK1R	144 1
47	M13F	AK1R	146 1-6
48	M13F	AK1R	146 1-4
49	M13F	AK1R	146 2-5
50	M13F	AK1R	146 2-7
51	M13F	AK1R	142 2-2
52	M13F	AK1R	142 1-1
53	M13F	AK1R	142 2-7
54	M13F	AK1R	142 2-3
55	M13F	AK1R	145 2-11
56	M13F	AK1R	145 1-17
57	M13F	AK1R	145 2-9
58	M13F	AK1R	145 2-17
59	M13F	AK1R	143 5
60	M13F	AK1R	143 17
61	M13F	AK1R	143 15
62	M13F	AK1R	143 21

PCR Polymerase : 2*KAPA hifi robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/24

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	141 2-1
2	M13F	M13R	141 2-2
3	M13F	M13R	141 2-4
4	M13F	M13R	141 2-6
5	M13F	M13R	142 1-5
6	M13F	M13R	142 2-4
7	M13F	M13R	142 1-3
8	M13F	M13R	142 1-4
9	M13F	M13R	143 13
10	M13F	M13R	143 19
11	M13F	M13R	143 25
12	M13F	M13R	144 18
13	M13F	M13R	144 17
14	M13F	M13R	144 5
15	M13F	M13R	144 9
16	M13F	M13R	143 19
17	M13F	M13R	145 1-5
18	M13F	M13R	145 2-15
19	M13F	M13R	143 2-13
20	M13F	M13R	145 1-13
21	M13F	AK1R	141 2-1
22	M13F	AK1R	141 2-2
23	M13F	AK1R	141 2-4
24	M13F	AK1R	141 2-6
25	M13F	AK1R	142 1-5
26	M13F	AK1R	142 2-4
27	M13F	AK1R	142 1-3
28	M13F	AK1R	142 1-4
29	M13F	AK1R	145 1-5
30	M13F	AK1R	145 2-15
31	M13F	AK1R	143 2-13
32	M13F	AK1R	145 1-13
33	M13F	AK1R	144 17
34	M13F	AK1R	144 5
35	M13F	AK1R	144 9
36	M13F	AK1R	143 19
37	M13F	AK1R	143 13
38	M13F	AK1R	143 19
39	M13F	AK1R	143 25
40	M13F	AK1R	144 9

PCR Polymerase : 2*KAPA Robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/26

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK27	IK30	GPDp + Rev after gel extraction
2	IK27	IK30	GPDp + Rev after gel extraction
3	IK27	IK30	GPDp + Rev after gel extraction
4	IK27	IK30	GPDp + Rev after gel extraction
5	IK23	IK28	GPDt after PCR purification
6	IK23	IK28	GPDt after PCR purification
7	IK23	IK28	GPDt after PCR purification
8	IK23	IK28	GPDt after PCR purification

PCR Polymerase : 2*KAPA hifi HS Readymix
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/26

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	145-2 11
2	M13F	M13R	145-1 9
3	M13F	M13R	145 2-7
4	M13F	M13R	145 1-21
5	M13F	M13R	145 1-3
6	M13F	M13R	145 2-3
7	M13F	M13R	145 1-19
8	M13F	M13R	142 1-2
9	M13F	AK1F	145-2 11
10	M13F	AK1F	145-1 9
11	M13F	AK1F	145 2-7
12	M13F	AK1F	145 1-21
13	M13F	AK1F	145 1-3
14	M13F	AK1F	145 2-3
15	M13F	AK1F	145 1-19
16	M13F	AK1F	142 1-2

PCR Polymerase : 2*KAPA hifi
98.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 25sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle

08/26

Restriction Enzyme digestion

Ohara

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	114-1	2.0	Pst1	1.0	10xH	1.0	10.0
2	114-2	2.0	Pst1	1.0	10xH	1.0	10.0
3	172	2.0	Pst1	1.0	10xH	1.0	10.0
4	172-1	2.0	Pst1	1.0	10xH	1.0	10.0
5	172-2	2.0	Pst1	1.0	10xH	1.0	10.0
6	172-4	2.0	Pst1	1.0	10xH	1.0	10.0
7	175-3	2.0	Pst1	1.0	10xH	1.0	10.0
8	175-10	2.0	Pst1	1.0	10xH	1.0	10.0
9	176	2.0	Pst1	1.0	10xH	1.0	10.0
10	177	2.0	Pst1	1.0	10xH	1.0	10.0

08/27

Nebuilder

Yoshimoto

Sample	Vectorname	Vector amount (ul)	Insert1 name	Insert1 amount(ul)	Insert2 name	Insert2 amount (ul)	Mastermix amount (ul)	Total amount(ul)
1	Rev	3.02	GPDp+Rev	0.669	GPDt	0.875	10.0	Total amount(ul)
2	173.0	3.02	164.0	0.325	165.0	0.285	5.0	Total amount(ul)
3	175.0	3.02	161.0	0.19	167.0	0.42	5.0	Total amount(ul)
4	178.0	3.02	163.0	0.41	167.0	0.42	5.0	Total amount(ul)

08/27

Restriction Enzyme digestion

Ohara

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	171-1	5.0	Pst1	1.0	10xH	1.0	10.0
2	171-1-2	5.0	Pst1	1.0	10xH	1.0	10.0
3	172-2	5.0	Pst1	1.0	10xH	1.0	10.0
4	172-2-2	5.0	Pst1	1.0	10xH	1.0	10.0
5	172-3	5.0	Pst1	1.0	10xH	1.0	10.0
6	172-4	5.0	Pst1	1.0	10xH	1.0	10.0
7	172-5	5.0	Pst1	1.0	10xH	1.0	10.0
8	172-6	5.0	Pst1	1.0	10xH	1.0	10.0
9	172-8	5.0	Pst1	1.0	10xH	1.0	10.0
10	172-9	5.0	Pst1	1.0	10xH	1.0	10.0
11	172-10	5.0	Pst1	1.0	10xH	1.0	10.0
12	172-11	5.0	Pst1	1.0	10xH	1.0	10.0
13	172-12	5.0	Pst1	1.0	10xH	1.0	10.0
14	172-13	5.0	Pst1	1.0	10xH	1.0	10.0
15	175-1	5.0	EcoR1	1.0	10xH	1.0	10.0
16	175-2	5.0	EcoR1	1.0	10xH	1.0	10.0
17	175-3	5.0	EcoR1	1.0	10xH	1.0	10.0
18	175-4	5.0	EcoR1	1.0	10xH	1.0	10.0
19	176-1	5.0	EcoR1	1.0	10xH	1.0	10.0
20	177-2	5.0	EcoR1	1.0	10xH	1.0	10.0
21	177-3	5.0	EcoR1	1.0	10xH	1.0	10.0
22	177-4	5.0	EcoR1	1.0	10xH	1.0	10.0
23	177-5	5.0	EcoR1	1.0	10xH	1.0	10.0
24	177-1	5.0	EcoR1	1.0	10xH	1.0	10.0
25	177-1-2	5.0	EcoR1	1.0	10xH	1.0	10.0
26	171-1	5.0	EcoR1	1.0	10xH	1.0	10.0
27	172-2	5.0	EcoR1	1.0	10xH	1.0	10.0
28	175-1	5.0	EcoR1	1.0	10xH	1.0	10.0
29	175-2	5.0	EcoR1	1.0	10xH	1.0	10.0
30	175-3	5.0	EcoR1	1.0	10xH	1.0	10.0
31	175-4	5.0	EcoR1	1.0	10xH	1.0	10.0
32	176-1	5.0	EcoR1	1.0	10xH	1.0	10.0
33	176-2	5.0	EcoR1	1.0	10xH	1.0	10.0
34	177-2	5.0	EcoR1	1.0	10xH	1.0	10.0
35	177-3	5.0	EcoR1	1.0	10xH	1.0	10.0
36	177-4	5.0	EcoR1	1.0	10xH	1.0	10.0
37	177-5	5.0	EcoR1	1.0	10xH	1.0	10.0

08/27

Restriction Enzyme digestion

Ohara

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	171 1-1	5.0	Pst1	1.0	10xH	1.0	10.0
2	171 1-2	5.0	Pst1	1.0	10xH	1.0	10.0
3	171 2	5.0	Pst1	1.0	10xH	1.0	10.0
4	171 3	5.0	Pst1	1.0	10xH	1.0	10.0
5	172-1	5.0	Pst1	1.0	10xH	1.0	10.0
6	172-2	5.0	Pst1	1.0	10xH	1.0	10.0
7	172-3	5.0	Pst1	1.0	10xH	1.0	10.0
8	172-4-1	5.0	Pst1	1.0	10xH	1.0	10.0
9	172-4-2	5.0	Pst1	1.0	10xH	1.0	10.0
10	172-5	5.0	Pst1	1.0	10xH	1.0	10.0
11	172-6	5.0	Pst1	1.0	10xH	1.0	10.0
12	172-8	5.0	Pst1	1.0	10xH	1.0	10.0
13	172-9	5.0	Pst1	1.0	10xH	1.0	10.0
14	172-10	5.0	Pst1	1.0	10xH	1.0	10.0
15	172-11	5.0	Pst1	1.0	10xH	1.0	10.0
16	172-12	5.0	Pst1	1.0	10xH	1.0	10.0
17	172-13	5.0	Pst1	1.0	10xH	1.0	10.0
18	171-1-1	5.0	EcoR1	1.0	10xH	1.0	10.0
19	174-1	5.0	EcoR1	1.0	10xH	1.0	10.0
20	175-1	5.0	EcoR1	1.0	10xH	1.0	10.0
21	175-2	5.0	EcoR1	1.0	10xH	1.0	10.0
22	175-3	5.0	EcoR1	1.0	10xH	1.0	10.0
23	175-4	5.0	EcoR1	1.0	10xH	1.0	10.0
24	176-1	5.0	EcoR1	1.0	10xH	1.0	10.0
25	176-2	5.0	EcoR1	1.0	10xH	1.0	10.0
26	177-2	5.0	EcoR1	1.0	10xH	1.0	10.0
27	177-3	5.0	EcoR1	1.0	10xH	1.0	10.0
28	177-4	5.0	EcoR1	1.0	10xH	1.0	10.0
29	177-5	5.0	EcoR1	1.0	10xH	1.0	10.0

08/28

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	145-2	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
2	142 2-11	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
3	142 2-2	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
4	142 2-1	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
5	142 2-3	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
6	242 1-4	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
7	142 2-4	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
8	142 1-5	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
9	142 1-3	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
10	142 2-5	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
11	142 1-2	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
12	142 1-1	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
13	145 2-3	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
14	145 1-2	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
15	145 2-7	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
16	145 4-9	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
17	145 1-11	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
18	145 1-3	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
19	145 2-11	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
20	145 2-5	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
21	145 2-8	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
22	145 1-14	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
23	145 2-15	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
24	145 2- 4-2	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
25	145 1-17	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
26	145 2-17	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0

08/29

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	142 1-3	5.0	EcoR1	0.5	Not1	0.5	10xH, BSA	1ul 1ul	10.0
2	145 1-11	5.0	Xba1	0.5	Not1	0.5	10xK, BSA	1ul 1ul	9.0
3	145 1-3	5.0	Xba1	0.5	Not1	0.5	10xK, BSA	1ul 1ul	9.0
4	145 2-42	5.0	Xba1	0.5	Not1	0.5	10xK, BSA	1ul 1ul	9.0

08/29

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK27	IK28	Rev, GPDp, GPDt
2	IK27	IK28	Rev+GPDp, GPDt
3	IK27	IK28	Rev plasmid
4	IK27	IK28	Rev plasmid
5	IK27	IK28	Rev plasmid
6	IK27	IK28	Rev plasmid

PCR Polymerase : KAKA Robust
95.0°C : 3min
98.0°C : 20sec
60.0°C : 15sec
72.0°C : 1min30sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

08/31

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	141 1-2	3.5	EcoR1	0.5	Xba1	0.5	10xN	1.0	10.0
2	141 1-2	3.5	EcoR1	0.5	Xba1	0.5	10xN	1.0	10.0
3	141 1-3	3.5	EcoR1	0.5	Xba1	0.5	10xN	1.0	10.0
4	141 1-3	3.5	EcoR1	0.5	Xba1	0.5	10xN	1.0	10.0
5	144-9	3.5	EcoR1	0.5	Not1	0.5	10xH, BSA	1ul, 1ul	10.0
6	144-9	3.5	EcoR1	0.5	Not1	0.5	10xH, BSA	1ul, 1ul	10.0
7	144 17	3.5	EcoR1	0.5	Not1	0.5	10xH, BSA	1ul, 1ul	10.0
8	144 17	3.5	EcoR1	0.5	Not1	0.5	10xH, BSA	1ul, 1ul	10.0
9	144 3	3.5	EcoR1	0.5	Not1	0.5	10xH, BSA	1ul, 1ul	10.0
10	144 3	3.5	EcoR1	0.5	Not1	0.5	10xH, BSA	1ul, 1ul	10.0
11	144 3	3.5	EcoR1	0.5	Not1	0.5	10xH, BSA	1ul, 1ul	10.0
12	144 1	3.5	EcoR1	0.5	Not1	0.5	10xH, BSA	1ul, 1ul	10.0
13	144 1	3.5	EcoR1	0.5	Not1	0.5	10xH, BSA	1ul, 1ul	10.0
14	144 1	3.5	EcoR1	0.5	Not1	0.5	10xH, BSA	1ul, 1ul	10.0
15	143 21	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
16	143 21	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
17	143 21	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
18	143 15	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
19	143 15	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
20	143 15	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
21	143 15	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
22	143 5	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
23	143 5	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
24	143 5	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
25	143 13	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
26	145 1-3	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
27	145 1-3	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
28	145 1-3	3.5	Xba1	0.5	Not1	0.5	10xK, BSA	1ul, 1ul	10.0
29	142 2-2	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
30	142 1-3	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
31	142	5.0	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
32	142 2-2	5.0	Not1	0.5	Xba1	0.5	10xK BSA	1ul, 1ul	10.0
33	142 1-3	5.0	Not1	0.5	Xba1	0.5	10xK BSA	1ul, 1ul	10.0
34	142	5.0	Not1	0.5	Xba1	0.5	10xK BSA	1ul, 1ul	10.0

09/01

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	P1
2	M13F	M13R	P2
3	M13F	M13R	P3
4	M13F	M13R	P4
5	M13F	M13R	T1
6	M13F	M13R	T2
7	M13F	M13R	T3
8	M13F	M13R	T4

PCR Polymerase : KAPA
98.0°C : 3min
98.0°C : 30sec
60.0°C : 20sec
72.0°C : 40sec
72.0°C : 10min
4.0°C : Infinate
X35 cycle

09/02

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	144-3	3.5	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
2	142	3.5	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
3	145 1-11	3.5	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0

09/02

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	165-1
2	M13F	M13R	165-2
3	M13F	M13R	165-3
4	M13F	M13R	165-4
5	M13F	IK60	165-1
6	M13F	IK60	165-2
7	M13F	IK60	165-3
8	M13F	IK60	165-4

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 30sec
60.0°C : 20sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/02

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	142 2-2	3.5	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
2	1421	3.5	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0
3	142 1-3	3.5	Xba1	0.5	EcoR1	0.5	10xM	1.0	10.0

09/03

Ligation

Yoshimoto

sample	Vector name	Vector amount(ul)	Insert name	Insert amount(ul)
1	YEplac195/sma1	0.35	161	6.19
2	YEplac195/sma1	0.35	162	12.2
3	YEplac195/sma1	0.35	163	13.1
4	YEplac195/sma1	0.35	164	10.32
5	YEplac195/sma1	0.35	166	17.25
6	YEplac195/sma1	0.35	167	13.28

09/03

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	IK55	GFPgalp-1
2	M13F	IK55	GFPgalp-2
3	M13F	IK55	GFPgalp-3
4	M13F	IK55	GFPgalp-4
5	M13F	IK55	GFPt-1
6	M13F	IK55	GFPt-2
7	M13F	IK55	GFPt-3
8	M13F	IK55	GFPt-4

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 30sec
60.0°C : 20sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/04

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK21	IK22	GPDp
2	IK21	IK22	GPDp
3	IK21	IK22	GPDp
4	IK21	IK22	GPDp
5	IK23	IK28	GPDter
6	IK23	IK28	GPDter
7	IK23	IK28	GPDter
8	IK23	IK28	GPDter
9	IK29	IK30	Rev long
10	IK29	IK30	Rev long
11	IK29	IK30	Rev long
12	IK29	IK30	Rev long
13	IK21	IK28	GPDter(PCRp), Rev(PCRp), GPDp(gel ex)
14	IK21	IK28	GPDter(PCRp), Rev+GPDp(PCRp)

PCR Polymerase : KAPA
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min20sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle
PCR pulification: 1~4
PCR pulification: 9~12
Gel extraction: 1~4
Gel extraction: 9~12

09/04

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK60	AK1F	142-2-1
2	IK60	AK1F	142-2-2
3	IK60	AK1F	142-2-3
4	IK60	AK1F	142-2-4
5	IK60	AK1F	142-2-5
6	IK60	AK1F	142-1-1
7	IK60	AK1F	142-1-2
8	IK60	AK1F	142-1-3
9	IK60	AK1F	142-1-4
10	IK60	AK1F	142-1-5
11	IK60	M13F	144-3
12	IK60	M13R	145-1-11
13	IK60	AK1F	144-3
14	IK60	AK1F	145-1-11

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 30sec
60.0°C : 20sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/04

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK60	M13R	142-2-1
2	IK60	M13R	142-2-2
3	IK60	M13R	142-2-3
4	IK60	M13R	142-2-4
5	IK60	M13R	142-2-5
6	IK60	M13R	142-1-1
7	IK60	M13R	142-1-2
8	IK60	M13R	142-1-3
9	IK60	M13R	142-1-4
10	IK60	M13R	142-1-5
11	M13F	M13R	gfpp
12	IK55	M13R	gfpp
13	M13F	M13R	gfpt M13
14	IK55	M13R	gfp 55

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 20sec
61.0°C : 20sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/04

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK27	IK30	Rev+p
2	IK21	IK30	Rev+p
3	IK27	IK22	GPDp
4	IK27	IK22	GPDp
5	IK27	IK22	GPDp
6	IK21	IK22	GPDp
7	IK21	IK22	GPDp
8	IK21	IK22	GPDp

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 30sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle

09/04

PCR

Fukuda

lane	Primer Foward	Primer Revese	Template
1	eef-1g F	eef-1gR	3
2	eef-1g F	eef-1gR	4
3	AK1F	AK1R	5
4	AK1F	AK1R	6
5	asbF	asbR	7
6	asbF	asbR	8

PCR Polymerase : KAPA Fast
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 12sec
72.0°C : 48sec
4.0°C : Infinate
X35 cycle

09/05

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	161
2	M13F	M13R	161
3	M13F	M13R	162
4	M13F	M13R	163
5	M13F	M13R	166
6	M13F	M13R	167
7	M13F	M13R	168
8	M13F	M13R	142
9	IK60	M13R	161
10	IK60	M13R	161
11	M13F	IK60	162
12	IK60	M13R	163
13	IK60	M13R	166
14	M13F	IK60	167
15	M13F	IK60	168
16	M13F	IK60	142

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 20sec
61.0°C : 20sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/05

Restriction Enzyme digestion

Yoshimto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	gfpp	5.0	EcoRI	2.0	None	None	H	1.0	10.0
2	gfpp	5.0	Not1	1.0	HindⅢ	1.0	K	1.0	10.0

09/05

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK21	IK28	Rev, p, t
2	IK21	IK28	Rev+p, t
3	IK27	IK28	Rev, p, t
4	IK27	IK28	Rev+p, t
5	IK21	IK24	Rev, p, t
6	IK21	IK24	Rev+p, t

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 15sec
55.0°C : 15sec
72.0°C : 30sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle

09/05

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK21	IK30	9/5 PCR p Rev,pro
2	IK21	IK30	9/5 PCR p Rev,pro
3	IK21	IK30	9/5 PCR p Rev,pro
4	IK21	IK30	9/5 PCR p Rev,pro
5	IK29	IK24	9/5 PCR p Rev,ter
6	IK29	IK24	9/5 PCR p Rev,ter
7	IK29	IK24	9/5 PCR p Rev,ter
8	IK29	IK24	9/5 PCR p Rev,ter

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 15sec
55.0°C : 15sec
72.0°C : 30sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle
PCR pulification: 5.0
Gel extraction: 5.0

09/05

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK21	IK22	GPD1promoter
2	IK21	IK22	GPD1promoter
3	IK21	IK22	GPD1promoter
4	IK21	IK22	GPD1promoter
5	IK21	IK22	GPD1promoter

PCR Polymerase : 2*KAPA Robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/05

PCR

Fukuda

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	9
2	M13F	M13R	10
3	14-3-3 zeta F	14-3-3 zeta R	9
4	14-3-3 zeta F	14-3-3 zeta R	10
5	tropomyosin F	tropomyosin R	11
6	tropomyosin F	tropomyosin R	12

PCR Polymerase : KAPA Fast
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 12sec
72.0°C : 48sec
4.0°C : Infinate
X35 cycle

09/05

PCR

Fukuda

lane	Primer Foward	Primer Revese	Template
1	tropomyosin F	tropomyosin R	11
2	tropomyosin F	tropomyosin R	11
3	tropomyosin F	tropomyosin R	11
4	tropomyosin F	tropomyosin R	12
5	tropomyosin F	tropomyosin R	12
6	tropomyosin F	tropomyosin R	12
7	14-3-3 pro F	14-3-3 pro R	13
8	14-3-3 pro F	14-3-3 pro R	13
9	14-3-3 pro F	14-3-3 pro R	13
10	14-3-3 pro F	14-3-3 pro R	14
11	14-3-3 pro F	14-3-3 pro R	14
12	14-3-3 pro F	14-3-3 pro R	14

PCR Polymerase : KAPA Fast
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 12sec
72.0°C : 48sec
4.0°C : Infinate
X35 cycle

09/05

PCR

Fukuda

lane	Primer Foward	Primer Revese	Template
1	tropomyosin F	tropomyosin R	11
2	tropomyosin F	tropomyosin R	11
3	tropomyosin F	tropomyosin R	11
4	tropomyosin F	tropomyosin R	12
5	tropomyosin F	tropomyosin R	12
6	tropomyosin F	tropomyosin R	12
7	eef-1g F	eef-1g R	4
8	AK1 F	AK1 R	5
9	M13F	M13R	9
10	M13F	M13R	9
11	M13F	M13R	9
12	M13F	M13R	9
13	M13F	M13R	10
14	M13F	M13R	10
15	M13F	M13R	10
16	M13F	M13R	10

PCR Polymerase : KAPA Fast
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 12sec
72.0°C : 48sec
4.0°C : Infinate
X35 cycle

09/06

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	gfpp	5.0	EcoRI	2.0	None	None	H	1.0	10.0
2	gfpp	5.0	Not1	1.0	HindⅢ	1.0	K	1.0	10.0

09/06

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	163-1
2	M13F	M13R	163-2
3	M13F	M13R	164
4	M13F	M13R	142

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 20sec
61.0°C : 20sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/06

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK29	IK24	Rev+t
2	IK29	IK24	Rev+t
3	IK29	IK24	Rev+t
4	IK29	IK24	Rev+t
5	IK21	IK24	Rev+t, pro PCRp
6	IK29	IK30	Rev7(plasmid)
7	IK29	IK30	Rev7(plasmid)
8	IK29	IK30	Rev7(plasmid)

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 15sec
55.0°C : 15sec
72.0°C : 30sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle

09/06

PCR

Dou

lane	Primer Foward	Primer Revese	Template
1	IK21	IK22	P1
2	IK21	IK22	P1
3	IK21	IK22	P1
4	IK21	IK22	P1

PCR Polymerase : 2X KAPA Robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/07

PCR

Dou

lane	Primer Foward	Primer Revese	Template
1	IK21	IK22	p+Rev
2	IK21	IK22	p+Rev

PCR Polymerase : 2X KAPA Robust
95.0°C : 3min
95.0°C : 15sec
55.0°C : 15sec
72.0°C : 1min
72.0°C : 1min
4.0°C : Infinate
X35 cycle

09/07

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK60	M13R	142 PCR
2	IK60	M13R	142-1
3	IK60	M13R	142-2
4	IK60	M13R	142-3
5	IK60	M13R	142-4
6	IK60	M13R	142-5

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/07

PCR

Fukuda

lane	Primer Foward	Primer Revese	Template
1	IK61	IK62	K733018
2	IK61	IK62	K731250

PCR Polymerase : KAPA Robust
95.0°C : 3min
95.0°C : 15sec
60.0°C : 15sec
72.0°C : 15sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle

09/07

PCR

Dou

lane	Primer Foward	Primer Revese	Template
1	IK21	IK22	P1
2	IK21	IK22	P1
3	IK21	IK22	P1

PCR Polymerase : 2X KAPA Robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/08

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	141	5.0	XbaI	1.0	NotI	0.5	K	1.0	10.0
2	144	5.0	XbaI	1.0	NotI	0.5	K	1.0	10.0
3	143	5.0	XbaI	1.0	NotI	0.5	K	1.0	10.0
4	145	5.0	XbaI	1.0	NotI	0.5	K	1.0	10.0

09/08

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK23	IK28	Rev3
2	IK23	IK28	Rev4
3	IK23	IK28	2(nega)
4	IK23	IK28	Rev new
5	IK29	IK30	Rev3
6	IK29	IK30	Rev4
7	IK29	IK30	2(nega)
8	IK29	IK30	Rev new

PCR Polymerase : KAPA Robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/08

PCR

Dou

lane	Primer Foward	Primer Revese	Template
1	IK21	IK22	p(blue font)
2	IK21	IK22	p(blue font)
3	IK21	IK22	p(blue font)
4	IK21	IK22	p(blue font)

PCR Polymerase : 2X KAPA Robust
98.0°C : 3min
98.0°C : 20sec
60.0°C : 30sec
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/08

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK23	IK24	99(nega)
2	IK23	IK24	101
3	IK23	IK24	Rev3
4	IK23	IK24	Rev4
5	IK23	IK24	Rev5
6	IK23	IK24	Rev6
7	IK23	IK24	T(posi)
8	IK29	IK30	99
9	IK29	IK30	Rev5
10	IK29	IK30	Rev6

PCR Polymerase : KAPA
95.0°C : 3min
95.0°C : 15sec
55.0°C : 15sec
72.0°C : 30sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle

09/08

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK21	IK24	all
2	IK21	IK24	all
3	IK21	IK22	promoter
4	IK21	IK22	promoter
5	IK21	IK22	promoter
6	IK21	IK22	promoter
7	IK29	IK30	Rev
8	IK29	IK30	Rev
9	IK29	IK30	nega 99
10	IK29	IK30	water
11	IK29	IK24	Rev+t
12	IK29	IK24	Rev+t
13	IK23	IK24	plasmid
14	IK23	IK24	nega 99
15	IK23	IK24	posi(T PCRp)
16	IK23	IK24	posi(T PCRp)
17	IK23	IK24	posi(T PCRp)
18	IK23	IK24	posi(T PCRp)
19	IK23	IK24	water

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
55.0°C : 1min
72.0°C : 1min
72.0°C : 5min
10.0°C : Infinate
X35 cycle

09/08

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK21	IK22	PCRp X10
2	IK21	IK22	PCRp X10
3	IK21	IK22	genome X10
4	IK21	IK22	genome
5	IK21	IK22	water
6	IK21	IK24	all
7	IK21	IK24	p(PCRp),Rev+t gel ex
8	IK21	IK24	8/20 PCRp,Rev+t gel ex
9	IK21	IK24	RA IK21X10
10	IK21	IK24	RA
11	IK21	IK24	water

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min
72.0°C : 5min
10.0°C : Infinate
X35 cycle

09/09

Ligation

Yoshimoto

sample	Vector name	Vector amount(ul)	Insert name	Insert amount(ul)
1	143	2	141.0	2.0
2	145	2	144.0	2.0
3	143	2	144.0	2.0

09/09

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	142	20.0	XbaI	4.0	NotI	2.0	K	4.0	40.0

09/09

Ligation

Yoshimoto

sample	Vector name	Vector amount(ul)	Insert name	Insert amount(ul)
1	323	0.34	GFPp(gal1)	12.5

09/10

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	142-3	5.0	XbaI	0.5	NotI	0.5	K	1.0	10.0
2	142-5	5.0	XbaI	0.5	NotI	0.5	K	1.0	10.0
3	161-1	5.0	XbaI	0.5	NotI	0.5	K	1.0	10.0
4	163-1	5.0	XbaI	0.5	NotI	0.5	K	1.0	10.0
5	164	5.0	XbaI	0.5	NotI	0.5	K	1.0	10.0
6	166	5.0	XbaI	0.5	NotI	0.5	K	1.0	10.0

09/14

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	IK60	162
2	IK29	IK24	Rev
3	IK40	IK55	GFP

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
58.0°C : 1min
72.0°C : 1min
72.0°C : 2min
10.0°C : Infinate
X35 cycle

09/14

Ligation

Yoshimoto

sample	Vector name	Vector amount(ul)	Insert name	Insert amount(ul)
1	YEplac195/sma1	0.34	163.0	13.1
2	YEplac195/sma1	0.34	166.0	17.25
3	143	2	141.0	2.0
4	145	2	144.0	2.0
5	143	2	144.0	2.0

09/14

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK21	IK22	genome
2	IK21	IK22	genome
3	IK121	IK122	genome
4	IK121	IK122	genome
5	IK129	IK130	Rev
6	IK129	IK130	Rev
7	IK129	IK130	nega 99
8	IK129	IK130	water
9	IK21	IK22	water
10	IK121	IK122	water

PCR Polymerase : 2X primestar
98.0°C : 3min
98.0°C : 10sec
55.0°C : 15sec
72.0°C : 5sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle
PCR pulification: 1,2
PCR pulification: 4,5
Gel extraction: 1,2
Gel extraction: 4,5

09/14

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK121	IK122	p
2	IK121	IK122	p
3	IK21	IK22	p
4	IK21	IK22	p
5	IK121	IK122	genome
6	IK121	IK122	genome
7	IK21	IK24	p1, Rev+t
8	IK21	IK24	p1, Rev+t
9	IK129	IK24	Rev new, t
10	IK21	IK130	p, Rev new
11	IK129	IK24	water
12	IK21	IK130	water

PCR Polymerase : prime star max
98.0°C : 3min
98.0°C : 10sec
55.0°C : 15sec
72.0°C : 5sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle

09/15

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK129	IK130	Rev
2	IK129	IK130	Rev
3	IK129	IK130	Rev
4	IK21	IK24	OP2+(Rev+t)
5	IK129	IK24	NR1+t
6	IK21	IK130	NR1+OP2

PCR Polymerase : primestar
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 1min30sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle

09/15

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK129	IK24	NR1+t
2	IK129	IK24	NR1+t
3	IK129	IK24	NR1+t
4	IK129	IK24	NR1+t
5	IK129	IK24	water
6	K129	IK130	rev
7	K129	IK130	rev

PCR Polymerase : primestar
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 1min30sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle
PCR pulification: 1~5
PCR pulification: 6,7
Gel extraction: 1~5
Gel extraction: 6,7

09/15

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK129	IK24	RT1
2	IK121	IK122	genome
3	IK121	IK122	genome
4	IK121	IK122	genome
5	IK21	IK24	RT1+OP1
6	IK21	IK24	RT1+OP2
7	IK121	IK24	RT1+NP1

PCR Polymerase : primestar
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 1min30sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle

09/15

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK121	IK24	RT1+NP1
2	IK121	IK24	RT1+NP1
3	IK121	IK24	RT1+NP1
4	IK121	IK24	RT1+NP1
5	IK129	IK24	RT1
6	IK121	IK24	water

PCR Polymerase : primestar
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 1min30sec
72.0°C : 3min
4.0°C : Infinate
X35 cycle
PCR pulification: 1~4
Gel extraction: 1~4

09/16

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK60	M13R	161-1
2	IK60	M13R	161-2
3	IK60	M13R	163-1
4	IK60	M13R	163-2
5	IK60	M13R	163-3
6	IK60	M13R	163-4
7	IK60	M13R	166-1
8	IK60	M13R	166-2
9	IK60	M13R	166-3
10	IK60	M13R	166-4
11	IK60	M13R	166-5
12	IK60	M13R	166-6
13	IK60	M13R	162-1
14	IK60	M13R	162-3
15	IK60	M13R	161-1
16	IK60	M13R	165-2
17	IK60	M13R	165-3
18	IK60	M13R	165-4
19	IK60	M13R	165-5
20	IK60	M13R	167-1
21	IK60	M13R	167-2
22	IK60	M13R	167-3
23	M13R	IK40	Gal1GFP 1
24	M13R	IK40	Gal1GFP 2
25	M13R	IK40	Gal1GFP 3
26	M13R	IK40	Gal1GFP 4

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
60.0°C : 1min
72.0°C : 2min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/16

Ligation

Ohara

sample	Vector name	Vector amount(ul)	Insert name	Insert amount(ul)
1	325	0.528	prt2	0.26
2	325	3.65	prt2	7.33

09/16

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK121	IK24	prt2
2	IK121	IK24	prt3
3	IK121	IK24	prt3
4	IK121	IK24	prt3
5	IK121	IK24	prt3
6	IK121	IK122	genome

PCR Polymerase : primestar
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 10sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle
PCR pulification: 2~5
Gel extraction: 2~5

09/16

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK60	M13R	142-5
2	IK60	M13R	142-5
3	M13F	IK60	143-1
4	M13F	IK60	143-2
5	M13F	IK60	143-3
6	M13F	IK60	145-1
7	M13F	IK60	145-2
8	M13F	IK60	145-3

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
60.0°C : 1min
72.0°C : 2min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/16

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	genome

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
60.0°C : 1min
72.0°C : 2min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/16

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK60	M13F	164-1
2	IK60	M13F	164-2
3	M13F	IK40	Gal1 GFP 1
4	M13F	IK40	Gal1 GFP 2
5	M13F	IK40	Gal1 GFP 3
6	M13F	IK40	Gal1 GFP 4

PCR Polymerase : go taq
95.0°C : 2min
95.0°C : 1min
60.0°C : 1min
72.0°C : 1min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/17

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK121'	IK24	N(blue colony)
2	IK121'	IK24	1(Rev white)
3	IK121'	IK24	2
4	IK121'	IK24	3
5	IK121'	IK24	prt3
6	IK121'	IK122'	genome

PCR Polymerase : prime star
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 10sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle

09/17

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	172-0
2	M13F	M13R	172-1
3	M13F	M13R	172-2
4	M13F	M13R	172-3
5	M13F	M13R	172-4
6	M13F	M13R	172-5
7	M13F	M13R	172-6
8	M13F	M13R	172-8
9	M13F	M13R	172-9
10	M13F	M13R	172-10
11	M13F	M13R	172-11
12	M13F	M13R	172-12
13	M13F	M13R	172-13
14	M13F	M13R	114-11
15	M13F	M13R	114-13
16	M13F	M13R	114-0
17	M13F	M13R	171-1-1
18	M13F	M13R	171-2
19	M13F	M13R	174-1
20	M13F	M13R	175-1
21	M13F	M13R	175-2
22	M13F	M13R	175-3
23	M13F	M13R	175-4
24	M13F	M13R	176-0
25	M13F	M13R	176-1
26	M13F	M13R	176-2
27	M13F	M13R	177-1
28	M13F	M13R	177-2
29	M13F	M13R	177-3
30	M13F	M13R	177-4
31	M13F	M13R	177-5
32	M13F	M13R	water
33	M13F	M13R	26(GPD AK1)

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/17

Ligation

Ohara

sample	Vector name	Vector amount(ul)	Insert name	Insert amount(ul)
1	325	0.528	prt4	11.7

09/18

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK40	IK47	114-0
2	IK55	IK40	171-1
3	IK40	IK41	172-1
4	IK60	AK1F	176-0
5	IK60	AK1F	177-1
6	IK60	AK1F	177-2

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
58.0°C : 1min
72.0°C : 50sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/18

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK60	M13R	104-1
2	IK60	M13R	104-2

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
60.0°C : 1min
72.0°C : 5min
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/19

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	PrimerF	PrimerR	162-1
2	PrimerF	PrimerR	162-1
3	PrimerF	PrimerR	162-1
4	PrimerF	PrimerR	162-1
5	PrimerF	PrimerR	Rev
6	PrimerF	PrimerR	Rev
7	PrimerF	PrimerR	Rev
8	PrimerF	PrimerR	Rev

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/19

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	Ik29	IK30	OR1-1
2	Ik29	IK30	OR1-2
3	Ik29	IK30	OR1-3
4	Ik29	IK30	OR1-4
5	Ik29	IK30	OR1 nega
6	Ik29	IK30	R2

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/19

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK60	M13R	162-1
2	IK60	M13R	162-2
3	IK60	M13R	162-3

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/19

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK89	IK90	EGFP
2	IK89	IK90	EGFP
3	IK84	IK85	REV
4	IK86	IK87	REV

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle
PCR pulification: lane1,2
PCR pulification: lane3
Gel extraction: lane1,2
Gel extraction: lane3

09/20

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK40	IK55	171
2	IK40	IK55	171
3	IK40	IK55	171
4	IK40	IK55	171
5	IK40	IK55	171
6	IK40	IK55	171
7	IK40	IK60	177
8	IK40	IK60	177
9	IK40	IK41	172
10	IK40	IK41	172
11	IK40	IK41	172
12	IK40	IK41	172
13	IK40	IK41	172
14	IK40	IK41	172
15	IK40	IK60	176
16	IK40	IK60	176
17	IK46	M13F	148
18	IK46	M13F	148
19	IK46	M13F	148
20	IK46	M13F	148
21	IK46	M13F	148

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/21

PCR

Yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	OR-1(REV plasmid)
2	M13F	M13R	OR-2
3	M13F	M13R	OR-3
4	M13F	M13R	OR-4
5	M13F	M13R	OR-5
6	IK23	Ik24	OR-1
7	IK23	Ik24	OR-2
8	IK23	Ik24	OR-3
9	IK23	Ik24	OR-4
10	IK23	Ik24	OR-5

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

09/22

PCR

Ohara

lane	Primer Foward	Primer Revese	Template
1	IK121	IK24	prt3
2	IK121	IK24	num99
3	IK121	IK24	OR1-1
4	IK121	IK24	OR1-2
5	IK121	IK24	OR1-3
6	IK121	IK24	OR1-5
7	IK121	IK24	OR1-4

PCR Polymerase : prime star max
98.0°C : 3min
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 10sec
72.0°C : 2min
4.0°C : Infinate
X35 cycle

09/22

PCR

Dou

lane	Primer Foward	Primer Revese	Template
1	IK89	IK90	EGFP2-1
2	IK89	IK90	EGFP2-2

PCR Polymerase : go taq
95.0°C : 2min15sec
95.0°C : 1min
55.0°C : 1min
72.0°C : 2min15sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

10/04

Restriction Enzyme digestion

yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	Template 5	5.0	Xba	0.025	EcoR1	0.025	M	1.0	10.0
2	Template 5	5.0	Not1	0.025	EcoR1	0.025	H	1.0	10.0
3	Template 5	5.0	Not1	0.025	Xba1	0.025	H	1.0	10.0

10/04

PCR

ohara

lane	Primer Foward	Primer Revese	Template
1	OD21	OD23	eGFP plasmid
2	OD21	OD23	eGFP plasmid
3	OD22	OD23	eGFP plasmid
4	OD22	OD23	eGFP plasmid
5	OD31	OD32	T7-RRE c
6	OD31	OD32	T7-RRE c

PCR Polymerase : prime star max
98.0°C : 10sec
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 10sec
72.0°C : 10sec
15.0°C : Infinate
X35 cycle

10/04

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	pSB1C3	15.0	Xba1	5.0	Spe1	5.0	M	12.0	100.0
2	pSB1C3	15.0	Xba1	5.0	Pst1	5.0	M	12.0	100.0

10/05

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	vf	vr	Colony1~20(191)
2	vf	vr	Colony21~40(192)
3	vf	vr	Colony41~60(193)
4	vf	vr	Colony61~80(194)

PCR Polymerase : prime star max
98.0°C : 10sec
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 10sec
72.0°C : 10sec
15.0°C : Infinate
X35 cycle

10/05

Restriction Enzyme digestion

yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	template	5.0	Pst1	0.05	10xH	1.0	10.0

10/06

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD41	OD42	Gal-GFP
2	OD41	OD43	Gal-GFP
3	OD41	OD44	Gal-GFP
4	IK61	OD52	Gal-GFP
5	OD53	OD54	Gal-GFP

PCR Polymerase : prime star max
98.0°C : 10sec
98.0°C : 10sec
55.0°C : 5sec
72.0°C : 10sec
72.0°C : 10sec
15.0°C : Infinate
X35 cycle

10/07

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK61	OD54	T7-RRE
2	IK61	OD54	fukuda GFP

PCR Polymerase : pfu
95.0°C : 2min
95.0°C : 1min
55.0°C : 30sec
72.0°C : 4min
72.0°C : 4min
15.0°C : Infinate
X40 cycle

10/07

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD11	OD12	GPD-gfp
2	OD11	OD13	GPD-gfp
3	OD41	OD42	Gal-GFP
4	OD41	OD43	Gal-GFP
5	OD41	OD44	Gal-GFP
6	OD31	OD32	T7-RRE
7	IK61	IK54	fukuda GFP + T7RRE
8	IK61	IK54	fukuda GFP + T7RRE

PCR Polymerase : pfu
95.0°C : 2min
95.0°C : 1min
55.0°C : 30sec
72.0°C : 4min
72.0°C : 4min
15.0°C : Infinate
X40 cycle

10/09

Restriction Enzyme digestion

yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	RE2 name	RE2 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	T7 RRE	15.0	Xba1	3.0	Pst1	3.0	M	3.0	30.0

10/09

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK61	IK54	GFP+RRE

PCR Polymerase : pfu
95.0°C : 2min
95.0°C : 1min
55.0°C : 30sec
72.0°C : 4min
72.0°C : 4min
15.0°C : Infinate
X40 cycle

10/09

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD41	OD42	Gal-gfp
2	OD41	OD44	Gal-gfp

PCR Polymerase : pfu
95.0°C : 2min
95.0°C : 1min
55.0°C : 30sec
72.0°C : 4min
72.0°C : 4min
15.0°C : Infinate
X35 cycle

10/12

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD61	OD12	TDH3-eGFP
2	OD62	OD12	TDH3-eGFP
3	OD63	OD12	TDH3-eGFP
4	OD61	OD13	TDH3-eGFP
5	OD62	OD13	TDH3-eGFP
6	OD63	OD13	TDH3-eGFP
7	OD73	IK62	GFP
8	OD45	OD42	GFP
9	OD45	OD43	GFP
10	OD45	OD44	GFP
11	IK61	OD74	GFP

PCR Polymerase : KAPA HiFi fast
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

10/12

Restriction Enzyme digestion

Yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	Template14	5.0	Xba1	1.0	M	1.0	10.0
2	Template100	5.0	Xba1	1.0	M	1.0	10.0

10/12

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD62	OD12	yeast genome
2	OD62	OD13	yeast genome
3	OD73	IK62	fukuda gfp
4	OD45	OD42	gal gfp
5	IK61	OD74	fukuda gfp

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

10/13

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD62	OD12	yeast genome
2	OD62	OD12	yeast genome
3	OD62	OD13	TDH3-eGFP
4	OD62	OD13	TDH3-eGFP
5	OD73	IK62	fukuda GFP
6	IK61	OD71	fukuda GFP
7	OD72	OD54	T7-RRE

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

10/14

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD62	OD12	yeast genome
2	OD62	OD13	yeast genome
3	OD45	OD42	gal1 GFP
4	IK61	OD74	fukuda GFP
5	OD73	IK62	fukuda GFP

PCR Polymerase : KAPA
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

10/14

PCR

ohara

lane	Primer Foward	Primer Revese	Template
1	IK103	IK104	165 gBlock
2	IK107	IK108	161 gBlock
3	IK101	IK102	161 gBlock
4	IK105	IK106	163 gBlock

PCR Polymerase : yoshimoto
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

10/14

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD45	OD42	gal1 GFP
2	IK61	OD74	fukuda GFP
3	OD73	IK62	fukuda GFP
4	IK61	OD54	fukuda GFP

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

10/14

Restriction Enzyme digestion

yoshimoto

lane	Sample DNA name	DNA amount (ul)	RE1 name	RE1 amount(ul)	Buffer name	Buffer amount(ul)	Total volume(ul)
1	template	15.0	Xba1	3.0	M	3.0	100.0
2	template	15.0	Xba1	5.0	M	3.0	100.0

10/14

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD31	OD32	T7+RRE
2	OD22	OD23	eGFP

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

10/15

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK61	OD54	6+7

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

10/15

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK85	IK86	rev
2	IK87	Ik88	rev

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle
PCR pulification: 1~2
Gel extraction: 1~2

10/15

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD31	OD32	T7+RRE
2	OD22	OD23	TDH3promoter

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

10/15

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD31	OD32	T7+RRE
2	OD22	OD23	TDH3promoter
3	OD45	OD42	Gal eGFP

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

10/15

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK105	Ik106	Gblock 163

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

10/15

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK105	IK108	Gal1 loop
2	IK101	Ik104	GFP loop

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X35 cycle

10/15

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK85	IK86	Rev parts
2	IK87	IK88	Rev parts
3	IK61	OD54	T7-RRE-GFP

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

10/17

PCR

yoshitomo

lane	Primer Foward	Primer Revese	Template
1	IK61	OD54	4
2	IK61	OD54	5
3	IK61	OD54	7

PCR Polymerase : KAPA hifi
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

10/17

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK87	IK88	Rev
2	IK105	IK108	Gal1-loop

PCR Polymerase :
95.0°C : 5min
98.0°C : 20sec
55.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

10/18

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK105	IK108	template1

PCR Polymerase :
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

10/18

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	IK107	IK108	Gal1pro-loop
2	IK105	IK106	Gal1pro-loop

PCR Polymerase :
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

10/19

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD22	OD23	GPD-loop
2	IK87	IK88	Gal1-loop
3	IK105	IK108	T7-RRE
4	IK101	IK104	TDH3-eGFP
5	OD31	OD32	Rev

PCR Polymerase :
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

10/20

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	M13F	M13R	Colony 1-10 (RRE)
2	M13F	M13R	Colony11-17 (TDH3-eGFP)
3	M13F	M13R	Colony 18-21 (Rev)
4	M13F	M13R	Colony 22-23 (GPDp-loop)
5	M13F	M13R	Colony24-28 (GPDp-loop)
6	M13F	M13R	Colony 29-32 (T7-RRE)
7	M13F	M13R	Colony 33-35 (TDH3-eGFP)
8	M13F	M13R	Colony 36-37 (GPDp-loop)
9	M13F	M13R	Colony 38-40 (Gal1-loop)
10	M13F	M13R	Colony 41-43 (Rev)
11	M13F	M13R	Colony 44 (GPDp-loop)

PCR Polymerase :
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

10/22

PCR

yoshimoto

lane	Primer Foward	Primer Revese	Template
1	OD22	OD23	TDH3 GFP
2	IK87	IK88	Rev
3	IK105	IK108	Gal1
4	IK101	IK104	GPD1
5	OD31	OD32	T7-RRE

PCR Polymerase :
95.0°C : 5min
98.0°C : 20sec
58.0°C : 15sec
72.0°C : 40sec
72.0°C : 5min
4.0°C : Infinate
X30 cycle

7/3

Restriction Enzyme Digestion

Tong, Tamukai

component	volume(&microl)
BSA	0.5
buffer(10xH)	5
EcoR1	0.5
Pst1	0.5
Dpn1	0.5
dH2O	18

add 4&microL linearized plasmid backbone (25ng/&microL)
add 4&microL Enzyme Master Mix
digest 37℃, 30min
add 8&microL NaOAc (pH5.2,3M)
add 16&microL 99.5% EtOh
Vortex 2sec
add 184&microL 99.5% EtOH
add 72&microL DW