Team:Kyoto/Notebook

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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

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

7/3

Restriction Enzyme Digestion

Tong, Tamukai

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

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

7/5

OE-PCR

Nambu, Shimazoe

sample	Primer Forward	volume (ul)	Primer Reverse	volume (ul)	template DNA	volume (ul)	Q5 HF 2xMasterMix (ul)	DW (ul)
1	Z007	1.25	Z009	1.25	Yeast genome	1.0	1.0	9.0
2	Z008	1.25	Z011	1.25	GFP	1.0	1.0	9.0
3	Z010	1.25	Z012	1.25	Yeast genome	1.0	1.0	9.0
4	Z007	1.25	Z014	1.25	Yeast genome	1.0	1.0	9.0
5	Z013	1.25	Z016	1.25	RFP	1.0	1.0	9.0
6	Z015	1.25	Z012	1.25	Yeast genome	1.0	1.0	9.0

7/6

OE-PCR

Nambu, Shimazoe

sample	Primer Forward	volume (ul)	Primer Reverse	volume (ul)	Template DNA	volume (ul)	Q5 HF2x Master Mix (ul)	DW (ul)
1	Z007	1.25	Z009	1.25	Yeast genome	1.0	12.5	9.0
2	Z010	1.25	Z012	1.25	Yeast genome	1.0	12.5	9.0
3	Z007	1.25	Z014	1.25	Yeast genome	1.0	12.5	9.0
4	Z015	1.25	Z012	1.25	Yeast genome	1.0	12.5	9.0
5*	Z007	1.25	Z009	1.25	p079	1.0	12.5	9.0
6*	Z010	1.25	Z012	1.25	p079	1.0	12.5	9.0
7*	Z007	1.25	Z014	1.25	p079	1.0	12.5	9.0
8*	Z015	1.25	Z012	1.25	p079	1.0	12.5	9.0

*sample5~8 x 2set

7/6

colony_PCR

Mangrin colony	DW (ul)	Q5 HF2x MasterMix (ul)	m13 Forward (ul)	m13 Reverse (ul)
1	2.0	2.5	0.25	0.25
2	2.0	2.5	0.25	0.25
3	2.0	2.5	0.25	0.25
4	2.0	2.5	0.25	0.25
5	2.0	2.5	0.25	0.25

Store PCR product at 4℃.

7/10

Restriction Enzyme Digestion

Tong, Tamukai

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

add 4, 8 μl (add two by two to sample)
add 4, 8μl Enzyme master mix (add two by two to sample)digest 37℃ 30min
70℃ 20min
add 1,1.6 μl NaOAc pH5.2, 3M(add two by two to sample)
20, 40μl (add two by two to sample) 99.5% EtOHVortex
preserve in -20℃

7/12

OE-PCR

Nambu

sample	KOD one PCR master mix (ul)	TDH promoter	volume (ul)	ORF	volume (ul)	CYC terminator	volume (ul)	DW (ul)
A	12.5	5	1.5	GFP	1.5	6	1.5	8.0
B	12.5	7	1.5	RFP	1.5	8	1.5	8.0
C	12.5	5	1.5	GFP	1.5	6	1.5	8.0
D	12.5	7	1.5	RFP	1.5	8	1.5	8.0

PCR
98℃　0:30
98℃* 0:10
45℃* 0:05
68℃* 0:05
68℃ 2:00
10℃
(* x 30 cycle)

7/31

Transformation & Blue-White Selection

Shimazoe

Mix 10μl of Competent Cell and 2μl G,R Ligation Product on ice
On ice for 30min
Heat Shock 42℃ for 45sec
On ice for 1,2 min
Add 100μl SOC medium and culture in 37℃ for 1h
Spread on a plate (50μl X-gal added)

8/1

Colony PCR

Tong

Component	Volume(μl)
DW	30
Q5	37.5
m13f	3.75
m13r	3.75
colony	Poke
Total	75

Add 0.6μl 10x Buffer to all sample (1~14)
Culture in 10mL

8/7

YPD

Tong

Component	(1L)	(900mL)	(600mL)
Peptone	20g	18g	12g
Yeast Extract	10g	9g	6g
Agar	15g	13.5g	9g
DW	xmL	810ml	480mL
Glucose+DW	20g+ymL (x+y=1000)	18g	12g

fluid culture
cultured 9 yeast strains in liquid YPD.
Put 15 ml of YPD medium in a 50 ml sterile tube
push the yeast with a white chip, put it in the tube
loosen the lid and put it in the shaker

8/8

OD measure

Tong

A	OD
562	0.38
561	0.40
564	0.36
554	0.33
555	0.38
547	0.30
557	0.32
559	0.39

For each 8 samples, transferred to a 1.5 ml tube　1mL each.
For soy sauce yeast only 15 ml was centrifuged, decantation was repeated and scaled down to 1 ml.

8/13

Ligation

Nambu

Number	1	2	3	4	5	6
vector	426	316	426	316	426	316
insert	Mangrin	Mangrin	ZrFPS1-2	ZrFPS1-2	McHKT2	McHKT2

Mix the following material (Contamination are shown in the table above)

Plasmid(vector): 0.5&microL
Insert: 1.0&microl
Ligation High: 1.5&microl

Put in Heatblock

8/13

YPD culture

Tong

NaCl 25%(400mL)	null
Tryptone	8g
Yeast Extract	4g
Agar	8g
Glucose	8g
NaCl	100g

Normal	null
Tryptone	4g
Yeast Extract	2g
Agar	4g
Glucose	4g

Make
25% NaCl YPD solid medium 400 mL
Normal YPD solid medium 200 mL
YPD liquid medium 500 mL

8/14

Transformation

Tamukai

Sample Name	Sample Volume (μl)	Competent Cells/(μl)
Mangrin	3	15
ZrFPS1-2	3	15
McHKT2	3	15

8/18

Colony PCR

Nambu

Component	Amount(μl)
DW	112
Q5	140
m13f	14
m13r	14
Colony	Poke
Total	280

PCR　　　　　　　Colony
98℃ 0:30　　　　　 number　　Backbone　　Insert
98℃* 0:10　　　　　301~306　　SmaCIP　　AtHKT1
57℃* 0:30　　　　　307~317　　SmaCIP　　SseNHX1
72℃* 1:00　　　　　318~326　　SmaCIP　　ZrFPs1-1st
72℃ 2:00　　　　　 327~337　　SmaCIP　　AtNHXS1
4℃ ∞　　　　　　　338~356　　SmaCIP　　 FgβF3
(*× 30 cycle)

8/18

Liquid Culture

Nambu

Material	Volume
LB	90ml
Amp	90ul

mix materials ( Finally concentration Amp 100 ug/ml )
put colony* into the liquid
(*colony number: 304,309,310,313,314,315,317,322,325,336,342,343,352,353,354)
put in incubator at 37℃ for 18 hours

8/18

Liquid Culture

Tong

Number	Material
547	WT
554	NHA1Δ
555	G19, ΔENA1-4
557	NHA1Δ, ENA1Δ
559	ENA1Δ
561	Z.roxii ATCC42981
562	Z.roxii CBS732
564	Z.roxii NBRC1876

37℃, 130rpm
prepared each two pieces of plates of 0,5,10,15,20,25% salt concentration and observed the yeasts.

8/19

Ligation

Nambu

Number	1	2	3	4	5	6
gBlock	Mangrin	ZrFPS1-2nd	McHKT2	AtHKT1	ZrFPS1-1st	AtNHXS1

mix the following material

SmaCIP (Backbone) : 1ul
gBlock (Insert) : 1ul
Ligation High : 2ul
(Total : 4ul)

putin HeatBlock overnight at 18℃

8/19

PCR

Nambu

sample	content (gBlock)	primer1	primer2
1	Mangrin	Z026	Z027
2	ZrFPS1_2nd	Z026	Z027
3	McHKT2	Z026	Z027
4	AtHKT1	Z026	Z027
5	ZrFPS1_1st	Z026	Z027
6	AtNHXS1	Z026	Z027
7	SseNHX1	Z026	Z027
8	AVP1_1st + AVP1_2nd	Z026	Z027

94℃ 2:00
98℃* 0:10
48℃ *0:30
68℃ *3:00
68℃ 0:10
10℃
* x 30 cycle

8/20

Ligation

Nambu

number	gBlock (Insert)	volume (ul)	SmaCIP (backbone) (ul)	Ligation High (ul)
7	SdrG	1	1	2
8	SdrG-Fg overlap	1	1	2
9	ZrGPD1	1	1	2

Put in Incubator overnight at 18℃.

8/20

Transformation

Nambu

Sample Name	Sample Volume (μl)	Competent Cells/(μl)	Medium
Mangrin	4	16	SOC Medium
ZrFPS1_2nd	4	16	SOC Medium
McHKT2	4	16	SOC Medium
AtHKT1	4	16	SOC Medium
ZrFPS1_1st	4	16	SOC Medium
AtNHXS1	4	16	SOC Medium

8/21

Restriction Enzyme Digestion

Shimazoe

plasmid	plasmid volume (ul)	DW (ul)	10x Fast digest buffer (ul)	EcoR1 (ul)	Pst1 (ul)	Total (ul)
17	2	14	2	1	1	20
19	1	14	2	1	1	20
26	2	14	2	1	1	20
110	2	14	2	1	1	20
120	2	14	2	1	1	20

8/21

Digest plasmid

Tong

plasmid	plasmid volume (ul)	DW (ul)	10x Fast Digest Buffer (ul)	EcoR1 (ul)	Pst1 (ul)
304	2	14	2	1	1
309	2	14	2	1	1
313	2	14	2	1	1
314	2	14	2	1	1
317	2	14	2	1	1
325	2	14	2	1	1
342	2	14	2	1	1
322	2	14	2	1	1
336	2	14	2	1	1
343	2	14	2	1	1
352	2	14	2	1	1
353	2	14	2	1	1
354	2	14	2	1	1

15min 37℃ incubate

8/25

OE-PCR

Shimazoe

plasmid	plasmid volume (ul)	DW (ul)	Q5 (ul)	primer1	primer1 volume (ul)	primer2	primer2 volume (ul)
38 (AVP1_1st)	0.5	3.5	5	Z064	0.5	Z026(p)	0.5
83 (AVP1_2nd)	0.5	3.5	5	Z065	0.5	Z027(p)	0.5

8/25

colony_PCR

Shimazoe

sample	Q5 (ul)	DW (ul)	primer m13f (ul)	primer m13r (ul)
38	2.5	2.0	0.25	0.25
83	2.5	2.0	0.25	0.25

colony PCR x 2 set

8/26

Liquid Culture

Nambu

sample	LB (ml)	Amp(100mg/ml) (ul)
505	50	50
506	50	50
507	50	50
508	50	50
516	50	50
522	50	50
530	50	50

Put them in incubator at 37℃ for 19 hours.

8/27

OE-PCR

Shimazoe

content	volume (ul)
DW	19
KOD one	25
primer Z026	1.5
prmer Z027	1.5
AVP1-1	1
AVP1-2	2

8/27

Miniprep_Nanodrop

Nambu

sample:505,506,507,508,516,522,530

8/28

PCR

Shimazoe

Component	Amount(μl)
DW	8.7
KOD	10
Z026℗	0.6
Z064	0.6
38(template)	0.1
Total	20

98℃* 10s
50℃* 5s
68℃* 20s
68℃ 1min
4℃ ∞
　(*× 40 cycle)

8/28

PCR

Shimazoe

Component	Amount(μl)
DW	8.7
KOD	10
Z027℗	0.6
Z065	0.6
83(template)	0.1
Total	20

PCR
98℃* 10s
50℃* 5s
68℃* 20s
68℃ 1min
4℃ ∞
　(*× 40 cycle)

8/28

OE-PCR

Shimazoe

Component	Amount(μl)
DW	20
Z026℗	1.5
Z027℗	1.5
KOD one	25
AVP1-1st	1
AVP1-2nd	1
Total	50

PCR
98℃* 10s
65℃* 5s
68℃* 30s
68℃ 1min
4℃ ∞
　(*× 25 cycle )

8/28

Colony PCR

Shimazoe

Component	Amount(μl)
2× Gflex PCR Buffer	162.5
primer m13f	6.5
primer m13r	6.5
Tks Gflex DNA polymerase	6.5
DW	143
Colony	Poke
Total	325

PCR
　　　　　　　　　 　　　　 　　　 number
94℃ 1min　　　　　　 SdrG　　　　 601~611
98℃* 10s　　　　　　Sdr-Fg　　　　 612~623
55℃* 15s　　　　　　ZrGPD1　　　 624~631
68℃* 2min　　　　　 Mangrin　　　 633~638
4℃ ∞ 　　　　　　　Zr-FPS1-2nd 　　 639~642
　(*× 30 cycle)　　　　McHKT2 　　　643~649
　　　　　　　　　　ZrFPS1-1st 　　 650~657

8/28

Liquid Culture

Tong

Number	Material
547	WT
554	NHA1Δ
555	G19, ΔENA1-4
557	NHA1Δ, ENA1Δ
559	ENA1Δ
561	Z.roxii ATCC42981
562	Z.roxii CBS732
564	Z.roxii NBRC1876

8/28

YPD culture, LB culture

Tong

	100ml 9% YPD	50ml YPD	250ml LB
Peptone(g)	2	1	2.5
Yeast Extract(g)	1	0.5	1.25
Agar(g)	1.5	0.75	3.75
DW(ml)	100	50	250
Glucose(g)	2	1	×
NaCl(g)	9	×	1.25

8/29

PCR

Shimazoe

Component	Amount(μl)
5× Q5 Reaction Buffer	15
10mM dNTPs	1.5
10uM Z041	3.75
10uM Z042	3.75
Template	Poke
Q5 DNA Polymerase	0.75
DW	47.25
Total	72

Template
AtHKT1(304), SseNHXS1(317), AtNHXS1(336)

PCR
98℃ 30s
98℃* 10s
65℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)

8/29

PCR

Shimazoe

Component	Amount(μl)
5× Q5 Reaction Buffer	5
10mM dNTPs	0.5
10uM Z041	1.25
10uM Z042	1.25
Template	Poke
Q5 DNA Polymerase	0.25
DW	16.75
Total	24

Template
FgβF3

PCR
98℃ 30s
98℃* 10s
65℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)

8/30

Reaction Enzyme Digestion

Shimazoe

plasmid	plasmid(ul)	10× T(ul)	10× BSA(ul)	SmaⅠ(ul)	CIAP(ul)	DW(ul)	Total/(ul)
426	10	10	10	4	2	64	100
316	10	10	10	4	2	64	100
425	10	10	10	4	2	64	100
315	10	10	10	4	2	64	100
423	10	10	10	4	2	64	100
313	10	10	10	4	2	64	100

6 sample
37℃ overnight

8/30

OE-PCR

Shimazoe

	sample	volume (ul)	5× Q5 Reaction Buffer (ul)	10uM dNTPs (ul)	10uM Z026℗ (ul)	10uM Z027℗ (ul)	Z026-Z039 (ul)	Z040-Z027 (ul)	Q5 DNA Polymerase (ul)	DW (ul)	Total (ul)
A	AtHKT1	0.5	2	0.2	0.5	0.5	0.5	0.5	0.1	5.2	10
B	SseNHX1	0.5	2	0.2	0.5	0.5	0.5	0.5	0.1	5.2	10
C	AtNHXS1	0.5	2	0.2	0.5	0.5	0.5	0.5	0.1	5.2	10

PCR

98℃ 30s
98℃* 10s
72℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 25 cycle)

8/30

PCR

Shimazoe

template	volume (ul)	primer①	volume (ul)	primer②	volume (ul)	5× Q5 Reaction Buffer (ul)	10uM dNTPs (ul)	Q5 DNA Polymerase (ul)	DW (ul)	Total (ul)
AVP1-1st	0.5	10uM Z026℗	0.5	10uM Z064	0.5	2	0.2	0.1	6.2	10
AVP1-2nd	0.5	10uM Z027℗	0.5	10uM Z065	0.5	2	0.2	0.1	6.2	10

PCR
98℃ 30s
98℃* 10s
67℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 25 cycle)

8/30

OE-PCR

Shimazoe

number	template	volume (ul)	10uM Z026℗	10uM Z027℗	Z026-Z039 (ul)	Z040-Z027 (ul)	5× Q5 Reaction Buffer (ul)	10uM dNTPs (ul)	Q5 DNA Polymerase (ul)	DW (ul)	Total (ul)
A	AtHKT1	1	0.5	0.5	1	1	2	0.2	0.1	3.7	10
B	SseNHX1	1	0.5	0.5	1	1	2	0.2	0.1	3.7	10
C	AtNHXS1	1	0.5	0.5	1	1	2	0.2	0.1	3.7	10
D	GFP	1	0.5	0.5	1	1	2	0.2	0.1	3.7	10
E	RFP	1	0.5	0.5	1	1	2	0.2	0.1	3.7	10

PCR
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)

8/30

PCR

Shimazoe

template	volume (ul)	10uM primer①	volume (ul)	10uM primer②	volume (ul)	5× Q5 Reaction Buffer (ul)	10uM dNTPs (ul)	Q5 DNA Polymerase (ul)	DW (ul)	Total (ul)
AVP1-1st (PCR products)	1	Z026℗	1.25	Z064	1.25	5	0.5	0.25	15.75	25
AVP1-1st (Plasmid)	1	Z026℗	1.25	Z064	1.25	5	0.5	0.25	15.75	25
AVP1-2nd (PCR products)	1	Z027℗	1.25	Z065	1.25	5	0.5	0.25	15.75	25
AVP1-2nd (Plasmid)	1	Z027℗	1.25	Z065	1.25	5	0.5	0.25	15.75	25

PCR
98℃ 30s
98℃* 10s
65℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)

8/30

Yeast Selection

Tong

・add 3ul of cultures to 3ml of nutrient mediums
・measure OD
・concussion culture (30%, 180rpm)

8/31

OE-PCR

sample	template	volume (ul)	5× Q5 Reaction Buffer (ul)	10uM dNTPs (ul)	10uM Z026℗	10uM Z027℗	Z026-Z039 (ul)	Z040-Z027 (ul)	Q5 DNA Polymerase (ul)	DW (ul)	Total (ul)
A	AtHKT1	5	10	1	2.5	2.5	5	5	0.5	18.5	50
B	AtNHXS1	5	10	1	2.5	2.5	5	5	0.5	18.5	50
C	GFP	5	10	1	2.5	2.5	5	5	0.5	18.5	50
D	RFP	5	10	1	2.5	2.5	5	5	0.5	18.5	50

PCR
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)

8/31

PCR

	template	volume (ul)	10uM primer①	volume (ul)	10uM primer②	volume (ul)	5× Q5 Reaction Buffer (ul)	10uM dNTPs (ul)	Q5 DNA Polymerase (ul)	DW (ul)	Total (ul)
1	AVP1-2nd (PCR products)	1	Z065	1.25	Z027℗	1.25	5	0.5	0.25	15.75	25
2	AVP1-2nd (Plasmid)	1	Z065	1.25	Z027℗	1.25	5	0.5	0.25	15.75	25
3	GFP (PCR2)	1	Z026℗	1.25	Z027℗	1.25	5	0.5	0.25	15.75	25
4	RFP (PCR5)	1	Z026℗	1.25	Z027℗	1.25	5	0.5	0.25	15.75	25

PCR
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)

8/31

OE-PCR

Shimazoe

sample	template	volume (ul)	10uM Z026℗ (ul)	10uM Z027℗ (ul)	Z026-Z039 (ul)	Z040-Z027 (ul)	KOD one (ul)	DW (ul)	Total (ul)
A	SseNHX1	1	0.3	0.3	1	1	5	1.4	10
B	GFP	1	0.3	0.3	1	1	5	1.4	10
C	RFP	1	0.3	0.3	1	1	5	1.4	10

PCR
98℃* 10s
50℃* 5s
68℃* 30s
68℃ 30s
(*× 30 cycle)

9/1

Ligation

Shimazoe

Number	1	2	3	4
vector	pRS 426	pRS 316	pRS 423	pRS 313
insert	AtHKT1	AtHKT1	AtNHXS1	AtNHXS1

Mix the following material (Contamination are shown in the table above)

Plasmid(vector): 1.0ul
Insert: 1.0ul
Ligation High: 2.0ul

Put in Heatblock

9/1

OE-PCR

Shimazoe

template	volume (ul)	10uM Z026℗ (ul)	10uM Z027℗ (ul)	Z026-Z039 (ul)	Z040-Z027 (ul)	Q5 (ul)	DW (ul)	Total (ul)
SseNHX1	5	1.5	1.5	5	5	25	7	50

PCR
98℃ 30s
98℃* 10s
50℃* 20s
72℃* 1min30s
72℃ 2min
4℃ ∞
(*× 30 cycle)

9/1

OE-PCR

Shimazoe

template	volume (ul)	10uM Z026℗ (ul)	10uM Z027℗ (ul)	5× Q5 Reaction Buffer (ul)	10uM dNTPs (ul)	Q5 DNA Polymerase (ul)	DW (ul)	Total (ul)
AVP1-1st	1	1.25	1.25	5	0.5	0.25	14.75	25
AVP1-2nd	1	1.25	1.25	5	0.5	0.25	14.75	25

PCR
98℃ 30s
98℃* 10s
55℃* 20s
72℃* 2min
72℃ 2min
(*× 30 cycle)

9/1

Colony PCR

Shimazoe

Material	Volume
2× Emerald amp MAX PCR Master Mix	375
10uM m13f	7.5
10uM m13r	7.5
DW	360
Total	750

98℃ 30s
98℃* 10s
60℃* 30s
72℃* 4min
72℃ 4min
4℃ ∞
(*× 30cycle)

9/2

Ligation

Tong

Material	Volume(ul)
SseNHX1 (template)	2
Vector (313, 423)	2
Ligation High	4
Total	8

9/2

Transformation

Tong

Ligation products(ul)	Competent Cells/(ul)	Total(ul)
8 (SseNHX1)	25	33

on ice 20min

9/2

PCR

Shimazoe

Material	Volume
DW	6.8
KODone	10
Z027	0.6
Z065	0.6
template	2
Total	20

98℃ 10s
98℃* 10s
50℃* 5s
68℃* 30s
68℃ 30s
(*× 30cycle)

9/2

PCR

Shimazoe

Material	Volume
5× Q5 Reaction Buffer	5
10uM dNTPs	0.5
10uM primer①	1.25
10uM primer②	1.25
template	1
Q5 DNA Polymerase	0.25
DW	15.75
Total	25

primer① (Z026/Z040)
primer② (Z039/Z027)

98℃ 30s
98℃* 10s
54℃* 20s
72℃* 30s
72℃ 2min
4℃ ∞
(*× 30 cycle)

9/2

OE-PCR

Shimazoe

Material	Volume
2× Emerald Amp MAX	12.25
10uM Z026℗	0.25
10uM Z027℗	0.25
AVP1-1st	1
AVP1-2nd	1
DW	10.25
Total	25

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 3m30s
72℃ 3m30s
4℃ ∞
(*× 30 cycle)

9/2

OE-PCR

Shimazoe

Material	Volume
5× Q5 Reaction Buffer	10
10uM Z026℗	2.5
10uM Z027℗	2.5
10uM dNTPs	1
template⓪	5
template①	5
template②	5
Q5 DNA Polymerase	0.5
DW	18.5
Total	50

template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)

9/3

Ligation

Tong

sample	volume(ul)	pRS316(ul)	Ligation High(ul)	Total(ul)
Mangrin	1	2	3	6
ZrFPS1-1st	1	2	3	6
ZrFPS1-2nd	1	2	3	6
ZrGPD1	1	2	3	6
McHKT2	1	2	3	6
SdrG	1	2	3	6
SdrG-Fgβ	1	2	3	6

18℃overnight

9/4

Transformation

Ligation products(ul)	Competent Cells/(ul)	Total(ul)
6	55	61

on ice　20min
42℃　45sec
on ice　3min
SOC　550
37℃　40min

9/4

PCR

Tong

Material	volume (ul)
DW	22.8
Template (pIEG-SSS)	1.6
Primer F (Z067)	2
Primer R (Z066)	2
DNA Pol (Q5)	0.4
Reaction buffer	8
dNTP mix	3.2
Total	40

98℃ 30s
98℃* 10s
48℃* 30s
72℃* 3min
72℃ 2min
4℃ ∞
(*× 30cycle)

9/4

OE-PCR

Shimazoe

Material	Volume
5× Q5 Reaction Buffer	10
10uM Z026℗	2.5
10uM Z027℗	2.5
10uM dNTPs	1
template⓪	5
template①	5
template②	5
Q5 DNA Polymerase	0.5
DW	18.5
Total	50

template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)

9/4

OE-PCR

Shimazoe

Material	Volume
5× Q5 Reaction Buffer	10
10uM Z026	2.5
10uM Z027	2.5
10uM dNTPs	1
template⓪	5
template①	5
template②	5
Q5 DNA Polymerase	0.5
DW	18.5
Total	50

template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)

9/4

Colony PCR

Shimazoe

Material	Volume(ul)
2× Emerald amp MAX PCR Master Mix	40
10uM m13f	0.8
10uM m13r	0.8
DW	38.4
Total	80

98℃ 30s
98℃* 10s
60℃* 30s
72℃* 4min
72℃ 4min
4℃ ∞
(*× 30cycle)

9/4

Ligation

Tong

number	sample	volume(ul)	vector	volume(ul)	Ligation High(ul)	Total(ul)
1	AtHKT1	1	316,426	2	3	6
2	AtNHXS1	1	423,313	2	3	6
3	SseNHX1	1	423,313	2	3	6

9/5

PCR

Tong

Material	volume (ul)
DW	22
Template (pIEG-SSS)	1
Primer F (Z067)	1
Primer R (Z066)	1
PrimeSTAR Max	25
Total	50

98℃* 10s
55℃* 15s
72℃* 35s
(*× 30cycle)

9/5

OE-PCR

Shimazoe

Material	Volume
5× Q5 Reaction Buffer	12
10uM Z026℗	3
10uM Z027℗	3
10uM dNTPs	1.2
template⓪	6
template①	6
template②	6
Q5 DNA Polymerase	0.6
DW	22.2
Total	60

template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)

9/5

Transformation

Shimazoe

	1	2	3	4	5	6
sample	AtHKT1	AtHKT1	AtNHXS1	AtNHXS1	SseNHX1	SseNHX1
vector	426	316	423	313	423	313
competent cell (ul)	20	30	20	30	20	30

9/5

PCR

Shimazoe

template	volume (ul)	10uM primer① (ul)	volume (ul)	10uM primer② (ul)	volume (ul)	5× Q5 Reaction Buffer (ul)	10uM dNTPs (ul)	Q5 DNA Polymerase (ul)	DW (ul)	Total (ul)
AVP1-1st	1	Z026	1.25	Z071	1.25	5	0.5	0.25	15.75	25
AVP1-2nd	1	Z027	1.25	Z072	1.25	5	0.5	0.25	15.75	25
AVP1-1st	1	Z026	1.25	Z070	1.25	5	0.5	0.25	15.75	25

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)

9/5

OE-PCR

Shimazoe

Material	Volume
5× Q5 Reaction Buffer	10
10uM Z026℗	2.5
10uM Z027℗	2.5
AVP1-1st	2
AVP1-2nd	2
10uM dNTPs	1
Q5 DNA Polymerase	0.5
DW	24.5
Total	50

template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)

9/5

Colony PCR

Tamukai

Material	Volume(ul)
2× Emerald amp MAX PCR Master Mix	180
10uM m13f	7.2
10uM m13r	7.2
DW	180
Total	373.4

98℃ 30s
98℃* 10s
60℃* 30s
72℃* 4min
72℃ 4min
4℃ ∞
(*× 30cycle)

9/5

Genome extracion

Tamukai

Material	volume (ul)
Proteinase K	1.2ul
Extraction Reagent	1.2ml

・Seperate into 17 microtube 150uL each
・incubate in 70℃ for 10min
・on ice for 5min
・Add Precipation Reagent 90ul, vortex, on ice 10min, remove supreme
・Add 80ul Phend-chloroform, vortex, max rpm (15,000rpm) 10min, remove supreme
・ -30℃

9/5

OE-PCR

Shimazoe

Material	Volume(ul)
2× Gflex PCR Buffer	25
10uM Z026℗	1
10uM Z027℗	1
template⓪	1
template①	1
template②	1
Tks Gflex DNA Polymerase	1
DW	19
Total	50

⓪(SseNHX1/RFP)
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
(*× 30 cycle)

9/5

Ligation

Shimazoe

insert	volume(ul)	vector	volume(ul)	Ligation High(ul)	Total(ul)
AtHKT1	2	316	2	4	8
AtHKT1	2	426	2	4	8
AtNHXS1	2	423	2	4	8
AtNHXS1	2	313	2	4	8
SseNHX1	2	423	2	4	8
SseNHX1	2	313	2	4	8

9/6

Transformation

Shimazoe

Ligation products(ul)	Competent Cells/(ul)	Total(ul)
8	30	38

9/6

Colony PCR

Shimazoe

Material	Volume(ul)
2× Emerald amp MAX PCR Master Mix	125
10uM m13f	2.5
10uM m13r	2.5
DW	120
Total	250

98℃ 30s
98℃* 10s
60℃* 30s
72℃* 2min
72℃ 2min
4℃ ∞
(*× 30cycle)

9/6

OE-PCR

Shimazoe

Material	Volume(ul)
2× Gflex PCR Buffer	25
10uM Z026℗	1
10uM Z027℗	1
AVP1-1st	1
AVP1-2nd	1
Tks Gflex DNA Polymerase	1
DW	20
Total	50

94℃ 1min
98℃* 10s
55℃* 15s
68℃* 2min
68℃ 2min
(*× 30 cycle)

9/6

PCR

Shimazoe

Material	Volume
5× Q5 Reaction Buffer	5
10uM primer①	1.25
10uM primer②	1.25
10uM dNTPs	0.5
template	1
Q5 DNA Polymerase	0.25
DW	15.75
Total	25

ZrGPD1(primer①②:Z041,Z042 template:506), SdrG(primer①②:Z044,Z045 template:752cp)

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)

9/7

Restriction Enzyme Digestion

Tong

plasmid	plasmid(ul)	10×K buffer(ul)	10×H buffer(ul)	10× BSA(ul)	EcoRⅠ(ul)	NcoⅠ(ul)	PstⅠ(ul)	DpnⅠ(ul)	DW(ul)	Total/(ul)
38	10	4	×	4	2	2	×	×	28	40
83	10	4	×	4	×	2	2	×	28	40
pSB1C3	20	×	4	×	2	×	2	2	10	40
YCplac111	2	×	4	×	2	×	2	×	30	40

9/8

Restriction Enzyme Digestion

Tong

	volume(ul)	10×H buffer(ul)	EcoRⅠ(ul)	PstⅠ(ul)	DW(ul)	Total/(ul)
DNA 404	5	4	2	2	27	40

9/8

Ligation

Tong

sample	volume(ul)	vector	volume(ul)	Ligation High(ul)	Total(ul)
AtHKT1	2	316	2	4	8
AtHKT1	2	426	2	4	8
AtNHXS1	2	423	2	4	8
AtNHXS1	2	313	2	4	8
SseNHX1	2	423	2	4	8
SseNHX1	2	313	2	4	8

9/8

OE-PCR

Tong

template	volume (ul)	10uM Z026℗ (ul)	10uM Z027℗ (ul)	5× Q5 Reaction Buffer (ul)	10uM dNTPs (ul)	promoter(ul)	terminator(ul)	Q5 DNA Polymerase (ul)	DW (ul)	Total (ul)
ZrGPD1 ORF	5	2.5	2.5	10	1	5	5	0.5	18.5	50

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1min30s
72℃ 2min
4℃ ∞
(*× 30cycle)

9/8

Transformation

Tong

Ligation products(ul)	Competent Cells/(ul)	Total(ul)
8	30	38

・on ice 10min
・42℃ 45sec
・+SOC 300ul
・37℃ 40min

9/8

OE-PCR

Tong

template①	volume (ul)	template②	volume (ul)	10uM Z026℗ (ul)	10uM Z027℗ (ul)	2× Prime Star (ul)	DW (ul)	Total (ul)
AVP1-1st	0.5	AVP1-2nd	0.5	1	1	10	7	20

98℃* 10s
55℃* 15s
72℃* 20s
(*× 30cycle)

9/8

PCR

Tong

Material	volume (ul)
DW	22
Template (pIEG-SSS)	1
Primer F (Z067)	1
Primer R (Z066)	1
PrimeSTAR Max	25
Total	50

98℃* 10s
55℃* 15s
72℃* 35s
(*× 30cycle)

9/8

Restriction Enzyme Digestion

Tong

	volume(ul)	10×M buffer(ul)	EcoRⅠ(ul)	XbaⅠ(ul)	DW(ul)	Total/(ul)
AVP1-1st (38)	20	4	2	2	12	40
YCplac111	2	4	2	2	30	40

9/8

Ligation

Tong

material	volume(ul)
AVP1-1st	1.5
AVP1-2nd	1.5
vector	1.0
Ligation High	4.5
Total	9.0

9/9

OE-PCR

Tong

template	volume (ul)	Stalk (ul)	Z044 (ul)	Z047 (ul)	2× Prime Star (ul)	DW (ul)	Total (ul)
SdrG, Fgβ	1	1	1	1	20	16	40

98℃* 10s
55℃* 5s
72℃* 20s
(*× 30cycle)

9/9

OE-PCR

Tong

template	volume (ul)	10uM Z026℗ (ul)	10uM Z027℗ (ul)	promoter(ul)	terminator(ul)	2× Prime Star (ul)	DW (ul)	Total (ul)
ZrGPD1	0.5	1	1	0.5	0.5	10	6.5	20

9/9

OE-PCR

Tong

template①	volume (ul)	template②	volume (ul)	10uM Z026℗ (ul)	10uM Z027℗ (ul)	2× Prime Star (ul)	DW (ul)	Total (ul)
AVP1-1st	0.5	AVP1-2nd	0.5	0.5	0.5	10	8	20

98℃* 10s
55℃* 5s
72℃* 15s
(*× 30cycle)

9/9

Ligation

Tong

Material	volume (ul)
AVP1-1st	3
YCplac111	3
Ligation high	6
Total	12

9/10

Transformation

Tong

Ligation products(ul)	Competent Cells/(ul)	Total(ul)
12	36	48

on ice 20min

9/10

Colony PCR

Tong

Material	Volume(ul)
2× Emerald amp MAX PCR Master Mix	70
10uM m13f	1.4
10uM m13r	1.4
DW	67.2
Total	140

98℃ 30s
98℃* 10s
60℃* 30s
72℃* 4min
72℃ 4min
4℃ ∞
(*× 30cycle)

9/10

Colony PCR

Tamukai

Material	Volume(ul)
2× Emerald amp MAX PCR Master Mix	400
10uM m13f	8
10uM m13r	8
DW	384
Total	800

98℃ 30s
98℃* 10s
60℃* 30s
72℃* 2min
72℃ 2min
4℃ ∞
(*× 30cycle)

9/10

OE-PCR

Shimazoe

Material	Volume(ul)
2× Gflex PCR Buffer	25
10uM Z026℗	1
10uM Z027℗	1
template⓪	1
template①	1
template②	1
Tks Gflex DNA Polymerase	1
DW	19
Total	50

ZrGPD1
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 30 cycle)

9/10

Colony PCR

Nambu

Material	Volume(ul)
2× Emerald amp MAX PCR Master Mix	35
10uM m13f	0.6
10uM m13r	0.6
DW	33.6
Total	70

98℃ 30s
98℃* 10s
58℃* 30s
72℃* 4min
72℃ 4min
4℃ ∞
(*× 30cycle)

9/10

Restriction Enzyme Digestion

Shimazoe

material	volume(μl)
EcoT14Ⅰ	25
10×H buffer	50
λDNA	71
DW	354
Total	500

9/11

Restriction Enzyme Digestion

Nambu

material	volume(μl)
Mangrin(pRS425)	10
BSA	4
10×H buffer(10xH)	4
Not1	2
TritonX-100	4
DW	16
Total	40

9/11

Restriction Enzyme Digestion

Nambu

material	volume(μl)
AVP1	5
BSA	2
10×H buffer(10xH)	2
EcoR1	1
Nco1	1
Pst1	1
DW	8
Total	20

9/11

OE-PCR

Shimazoe

Material	Volume(ul)
2× Gflex PCR Buffer	30
10uM Z026℗	1.2
10uM Z027℗	1.2
template⓪	1.2
template①	1.2
template②	1.2
Tks Gflex DNA Polymerase	1.2
DW	22.8
Total	60

ZrGPD1
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 30 cycle)

9/11

Ligation

Shimazoe

insert	volume(ul)	vector	volume(ul)	Ligation High(ul)	Total(ul)
GFP	1	426	1	2	4
GFP	1	316	1	2	4
RFP	1	426	1	2	4
RFP	1	316	1	2	4

9/11

PCR

Tamukai

Material	Volume(ul)
2×Emerald Amp buffer	25
Z003	0.5
Z004	0.5
DW	24
Total	50

NHA1
98℃ 2min
98℃* 30s
48℃* 30s
68℃* 3m30s
68℃ 5min
4℃ ∞
(*× 30 cycle)

9/11

PCR

Tamukai

material	volume (ul)
DW	47.8
5× Q5 Reaction Buffer	16
10uM dNTPs	6.4
Q5 DNA Polymerase	0.8
10uM Z005	4
10uM Z028	4
template(pFA6a-hphMX)	1
Total	80

ENA1Δ1,2,5

9/11

PCR

Tamukai

material	volume (ul)
DW	45.8
5× Q5 Reaction Buffer	16
10uM dNTPs	6.4
Q5 DNA Polymerase	0.8
10uM Z005	4
10uM Z028	4
template(yeast genome)	1
Total	80

NHA1

9/11

Ligation

Shimazoe

insert	volume(ul)	vector	volume(ul)	Ligation High(ul)	Total(ul)
AtHKT1	1	316	1	2	4
AtHKT1	1	426	1	2	4
AtNHXS1	1	423	1	2	4
AtNHXS1	1	313	1	2	4
SseNHX1	1	423	1	2	4
SseNHX1	1	313	1	2	4

9/12

Restriction Enzyme Digestion

Nambu

material	volume(μl)
CIAP	1
pRS426 or pRS425	2
BSA	4
10×H buffer(10xH)	4
Not1	2
TritonX-100	4
DW	23
Total	40

material	volume(μl)
AVP1(on pSB1C3)	5
BSA	2
10×H buffer(10xH)	2
Not1	1
TritonX-100	4
DW	8
Total	20