Difference between revisions of "Team:Kyoto/Notebook"

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</div>
 
</div>
 
<!------------ Table END ------------>
 
<!------------ Table END ------------>
 +
 +
 +
 +
<!-- Table Generated by KELN Author: Nakazato -->
 +
<div class="keln_container">
 +
<a name="1010" class = "kyoto-jump"><span class="keln_date"><h3>10/10</h3></span></a>
 +
<span class="keln_exp"><h4>Transformation</h4></span>
 +
<span class="keln_researcher">Tong</span>
 +
<table class="keln_table">
 +
<tr><th>Labeling</th><th>Yeast type</th><th>URA</th><th>LEU</th><th>HIS</th></tr>
 +
<tr><td>73
 +
</td><td>ΔENA1
 +
</td><td>910AtHKT1(426)
 +
</td><td>AVP1-1(425)
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>74
 +
</td><td>ΔENA1
 +
</td><td>911 AtHKT1(426)
 +
</td><td>AVP1-1(425)
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>75
 +
</td><td>ΔENA1
 +
</td><td>empty, pRS426
 +
</td><td>AVP1-1(425)
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>76
 +
</td><td>ΔENA1
 +
</td><td>empty, pRS426
 +
</td><td>AVP1-1(425)
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>77
 +
</td><td>ΔENA1
 +
</td><td>910 AtHKT1(426)
 +
</td><td>empty, pRS425
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>78
 +
</td><td>ΔENA1
 +
</td><td>911 AtHKT1(426)
 +
</td><td>empty, pRS425
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>79
 +
</td><td>ΔENA1
 +
</td><td>910 AtHKT1(426)
 +
</td><td>AVP1-1(425)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>80
 +
</td><td>ΔENA1
 +
</td><td>911 AtHKT1(426)
 +
</td><td>AVP1-1(425)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>81
 +
</td><td>ΔENA1
 +
</td><td>empty, pRS426
 +
</td><td>AVP1-1(425)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>82
 +
</td><td>ΔENA1
 +
</td><td>empty, pRS426
 +
</td><td>empty, pRS425
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>83
 +
</td><td>ΔENA1
 +
</td><td>empty, pRS426
 +
</td><td>empty, pRS425
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>84
 +
</td><td>ΔENA1
 +
</td><td>empty, pRS426
 +
</td><td>empty, pRS425
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>85
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>910 AtHKT1(426)
 +
</td><td>AVP1-1(425)
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>86
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>911 AtHKT1(426)
 +
</td><td>AVP1-1(425)
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>87
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>empty, pRS426
 +
</td><td>AVP1-1(425)
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>88
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>empty, pRS426
 +
</td><td>AVP1-1(425)
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>89
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>910 AtHKT1(426)
 +
</td><td>empty, pRS425
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>90
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>911 AtHKT1(426)
 +
</td><td>empty, pRS425
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>91
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>910 AtHKT1(426)
 +
</td><td>AVP1-1(425)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>92
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>911 AtHKT1(426)
 +
</td><td>AVP1-1(425)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>93
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>empty, pRS426
 +
</td><td>AVP1-1(425)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>94
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>empty, pRS426
 +
</td><td>empty, pRS425
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>95
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>empty, pRS426
 +
</td><td>empty, pRS425
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>96
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>empty, pRS426
 +
</td><td>empty, pRS425
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>97
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>NHA1
 +
</td><td>AVP1-1(425)
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>98
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>NHA1
 +
</td><td>AVP1-1(425)
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>99
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>NHA1
 +
</td><td>empty, pRS425
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>100
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>NHA1
 +
</td><td>empty, pRS425
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>101
 +
</td><td>ΔENA1
 +
</td><td>910 AtHKT1(426)
 +
</td><td>AVP1(YCplac111)
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>102
 +
</td><td>ΔENA1
 +
</td><td>911 AtHKT1(426)
 +
</td><td>AVP1(YCplac111)
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>103
 +
</td><td>ΔENA1
 +
</td><td>empty, pRS426
 +
</td><td>AVP1(YCplac111)
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>104
 +
</td><td>ΔENA1
 +
</td><td>empty, pRS426
 +
</td><td>AVP1(YCplac111)
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>105
 +
</td><td>ΔENA1
 +
</td><td>910 AtHKT1(426)
 +
</td><td>AVP1(YCplac111)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>106
 +
</td><td>ΔENA1
 +
</td><td>911 AtHKT1(426)
 +
</td><td>AVP1(YCplac111)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>107
 +
</td><td>ΔENA1
 +
</td><td>empty, pRS426
 +
</td><td>AVP1(YCplac111)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>108
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>910 AtHKT1(426)
 +
</td><td>AVP1(YCplac111)
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>109
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>911 AtHKT1(426)
 +
</td><td>AVP1(YCplac111)
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>110
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>empty, pRS426
 +
</td><td>AVP1(YCplac111)
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>111
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>empty, pRS426
 +
</td><td>AVP1(YCplac111)
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>112
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>910 AtHKT1(426)
 +
</td><td>AVP1(YCplac111)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>113
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>911 AtHKT1(426)
 +
</td><td>AVP1(YCplac111)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>114
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>empty, pRS426
 +
</td><td>AVP1(YCplac111)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>115
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>NHA1
 +
</td><td>AVP1(YCplac111)
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>116
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>NHA1
 +
</td><td>AVP1(YCplac111)
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>117
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>NHA1
 +
</td><td>AVP1(YCplac111)
 +
</td><td>938 AtNHXS1(423)
 +
</td></tr><tr><td>118
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>NHA1
 +
</td><td>AVP1(YCplac111)
 +
</td><td>1301 SseNHX1(423)
 +
</td></tr><tr><td>119
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>NHA1
 +
</td><td>AVP1(YCplac111)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>120
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>NHA1
 +
</td><td>AVP1(YCplac111)
 +
</td><td>empty, pRS423
 +
</td></tr><tr><td>121
 +
</td><td>ΔENA1,2,5ΔNHA1
 +
</td><td>NHA1
 +
</td><td>AVP1(YCplac111)
 +
</td><td>empty, pRS423
 +
</td></tr>
 +
</table>
 +
</div>
 +
<!------------ Table END ------------>
 +
  
  
 
<br><br><br><br><br>
 
<br><br><br><br><br>

Revision as of 14:22, 15 October 2018

July
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8 9 10 11 12 13 14
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22 23 24 25 26 27 28
29 30 31
August
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12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
September
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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
componentvolume(μl)
BSA0.5
buffer(10xH)5
EcoR10.5
Pst10.5
Dpn10.5
dH2O18

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
samplePrimer Forwardvolume (ul)Primer Reversevolume (ul)template DNAvolume (ul)Q5 HF 2xMasterMix (ul)DW (ul)
1Z0071.25Z0091.25Yeast genome1.01.09.0
2Z0081.25Z0111.25GFP1.01.09.0
3Z0101.25Z0121.25Yeast genome1.01.09.0
4Z0071.25Z0141.25Yeast genome1.01.09.0
5Z0131.25Z0161.25RFP1.01.09.0
6Z0151.25Z0121.25Yeast genome1.01.09.0

7/6

OE-PCR

Nambu, Shimazoe
samplePrimer Forwardvolume (ul)Primer Reversevolume (ul)Template DNAvolume (ul)Q5 HF2x Master Mix (ul)DW (ul)
1Z0071.25Z0091.25Yeast genome1.012.59.0
2Z0101.25Z0121.25Yeast genome1.012.59.0
3Z0071.25Z0141.25Yeast genome1.012.59.0
4Z0151.25Z0121.25Yeast genome1.012.59.0
5*Z0071.25Z0091.25p0791.012.59.0
6*Z0101.25Z0121.25p0791.012.59.0
7*Z0071.25Z0141.25p0791.012.59.0
8*Z0151.25Z0121.25p0791.012.59.0

*sample5~8 x 2set

7/6

colony_PCR

Mangrin colonyDW (ul)Q5 HF2x MasterMix (ul)m13 Forward (ul)m13 Reverse (ul)
12.02.50.250.25
22.02.50.250.25
32.02.50.250.25
42.02.50.250.25
52.02.50.250.25

Store PCR product at 4℃.

7/10

Restriction Enzyme Digestion

Tong, Tamukai
componentvolume(μl)
BSA0.5
buffer(10xH)5
EcoR10.5
Pst10.5
Dpn10.5
dH2O18

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
sampleKOD one PCR master mix (ul)TDH promotervolume (ul)ORFvolume (ul)CYC terminatorvolume (ul)DW (ul)
A12.551.5GFP1.561.58.0
B12.571.5RFP1.581.58.0
C12.551.5GFP1.561.58.0
D12.571.5RFP1.581.58.0

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
ComponentVolume(μl)
DW30
Q537.5
m13f3.75
m13r3.75
colonyPoke
Total75

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

8/7

YPD

Tong
Component(1L)(900mL)(600mL)
Peptone20g18g12g
Yeast Extract10g9g6g
Agar15g13.5g9g
DWxmL810ml480mL
Glucose+DW20g+ymL (x+y=1000)18g12g

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
AOD
5620.38
5610.40
5640.36
5540.33
5550.38
5470.30
5570.32
5590.39

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
Number123456
vector426316426316426316
insertMangrinMangrinZrFPS1-2ZrFPS1-2McHKT2McHKT2

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
Tryptone8g
Yeast Extract4g
Agar8g
Glucose8g
NaCl100g

Normalnull
Tryptone4g
Yeast Extract2g
Agar4g
Glucose4g

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

8/14

Transformation

Tamukai
Sample NameSample Volume (μl)Competent Cells/(μl)
Mangrin315
ZrFPS1-2315
McHKT2315

8/18

Colony PCR

Nambu
ComponentAmount(μl)
DW112
Q5140
m13f14
m13r14
ColonyPoke
Total280

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
MaterialVolume
LB90ml
Amp90ul

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
NumberMaterial
547WT
554NHA1Δ
555G19, ΔENA1-4
557NHA1Δ, ENA1Δ
559ENA1Δ
561Z.roxii ATCC42981
562Z.roxii CBS732
564Z.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
Number123456
gBlockMangrinZrFPS1-2ndMcHKT2AtHKT1ZrFPS1-1stAtNHXS1

mix the following material

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

putin HeatBlock overnight at 18℃

8/19

PCR

Nambu
samplecontent (gBlock)primer1primer2
1MangrinZ026Z027
2ZrFPS1_2ndZ026Z027
3McHKT2Z026Z027
4AtHKT1Z026Z027
5ZrFPS1_1stZ026Z027
6AtNHXS1Z026Z027
7SseNHX1Z026Z027
8AVP1_1st + AVP1_2ndZ026Z027

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

8/20

Ligation

Nambu
numbergBlock (Insert)volume (ul)SmaCIP (backbone) (ul)Ligation High (ul)
7SdrG112
8SdrG-Fg overlap112
9ZrGPD1112

Put in Incubator overnight at 18℃.

8/20

Transformation

Nambu
Sample NameSample Volume (μl)Competent Cells/(μl)Medium
Mangrin416SOC Medium
ZrFPS1_2nd416SOC Medium
McHKT2416SOC Medium
AtHKT1416SOC Medium
ZrFPS1_1st416SOC Medium
AtNHXS1416SOC Medium

8/21

Restriction Enzyme Digestion

Shimazoe
plasmid plasmid volume (ul)DW (ul)10x Fast digest buffer (ul)EcoR1 (ul)Pst1 (ul)Total (ul)
1721421120
1911421120
2621421120
11021421120
12021421120
0821Restriction Enzyme Digestion

8/21

Digest plasmid

Tong
plasmidplasmid volume (ul)DW (ul)10x Fast Digest Buffer (ul)EcoR1 (ul)Pst1 (ul)
304214211
309214211
313214211
314214211
317214211
325214211
342214211
322214211
336214211
343214211
352214211
353214211
354214211
0821Digest plasmid

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)0.53.55Z0640.5Z026(p)0.5
83 (AVP1_2nd)0.53.55Z0650.5Z027(p)0.5

8/25

colony_PCR

Shimazoe
sampleQ5 (ul)DW (ul)primer m13f (ul)primer m13r (ul)
382.52.00.250.25
832.52.00.250.25
0825colonyPCR1
0825colonyPCR2
0825colonyPCR3

colony PCR x 2 set

8/26

Liquid Culture

Nambu
sampleLB (ml)Amp(100mg/ml) (ul)
5055050
5065050
5075050
5085050
5165050
5225050
5305050

Put them in incubator at 37℃ for 19 hours.

8/27

OE-PCR

Shimazoe
contentvolume (ul)
DW19
KOD one25
primer Z0261.5
prmer Z0271.5
AVP1-11
AVP1-22

8/27

Miniprep_Nanodrop

Nambu

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

8/28

PCR

Shimazoe
ComponentAmount(μl)
DW8.7
KOD10
Z026℗0.6
Z0640.6
38(template)0.1
Total20
0828PCR

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

8/28

PCR

Shimazoe
ComponentAmount(μl)
DW8.7
KOD10
Z027℗0.6
Z0650.6
83(template)0.1
Total20

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

8/28

OE-PCR

Shimazoe
ComponentAmount(μl)
DW20
Z026℗1.5
Z027℗1.5
KOD one25
AVP1-1st1
AVP1-2nd1
Total50
0828OE-PCR

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

8/28

Colony PCR

Shimazoe
ComponentAmount(μl)
2× Gflex PCR Buffer162.5
primer m13f6.5
primer m13r6.5
Tks Gflex DNA polymerase6.5
DW143
ColonyPoke
Total325
0828ColonyPCR1
0828ColonyPCR2

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

8/28

YPD culture, LB culture

Tong
100ml 9% YPD50ml YPD250ml LB
Peptone(g)212.5
Yeast Extract(g)10.51.25
Agar(g)1.50.753.75
DW(ml)10050250
Glucose(g)21×
NaCl(g)9×1.25

8/29

PCR

Shimazoe
ComponentAmount(μl)
5× Q5 Reaction Buffer15
10mM dNTPs1.5
10uM Z0413.75
10uM Z0423.75
TemplatePoke
Q5 DNA Polymerase0.75
DW47.25
Total72

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
ComponentAmount(μl)
5× Q5 Reaction Buffer5
10mM dNTPs0.5
10uM Z0411.25
10uM Z0421.25
TemplatePoke
Q5 DNA Polymerase0.25
DW16.75
Total24
0829PCR2

Template
FgβF3

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

8/30

Reaction Enzyme Digestion

Shimazoe
plasmidplasmid(ul)10× T(ul)10× BSA(ul)SmaⅠ(ul)CIAP(ul)DW(ul)Total/(ul)
4261010104264100
3161010104264100
4251010104264100
3151010104264100
4231010104264100
3131010104264100

6 sample
37℃ overnight

8/30

OE-PCR

Shimazoe
samplevolume (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)
AAtHKT10.520.20.50.50.50.50.15.210
BSseNHX10.520.20.50.50.50.50.15.210
CAtNHXS10.520.20.50.50.50.50.15.210

PCR

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

8/30

PCR

Shimazoe
templatevolume (ul)primer①volume (ul)primer②volume (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
AVP1-1st0.510uM Z026℗0.510uM Z0640.520.20.16.210
AVP1-2nd0.510uM Z027℗0.510uM Z0650.520.20.16.210
0830PCR1

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

8/30

OE-PCR

Shimazoe
numbertemplatevolume (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)
AAtHKT110.50.51120.20.13.710
BSseNHX110.50.51120.20.13.710
CAtNHXS110.50.51120.20.13.710
DGFP10.50.51120.20.13.710
ERFP10.50.51120.20.13.710
0830OE-PCR2

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

8/30

PCR

Shimazoe
templatevolume (ul)10uM primer①volume (ul)10uM primer②volume (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
AVP1-1st (PCR products)1Z026℗1.25Z0641.2550.50.2515.7525
AVP1-1st (Plasmid)1Z026℗1.25Z0641.2550.50.2515.7525
AVP1-2nd (PCR products)1Z027℗1.25Z0651.2550.50.2515.7525
AVP1-2nd (Plasmid)1Z027℗1.25Z0651.2550.50.2515.7525
0830PCR2

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

sampletemplatevolume (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)
AAtHKT151012.52.5550.518.550
BAtNHXS151012.52.5550.518.550
CGFP51012.52.5550.518.550
DRFP51012.52.5550.518.550
0831OE-PCR1

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

8/31

PCR

templatevolume (ul)10uM primer①volume (ul)10uM primer②volume (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
1AVP1-2nd (PCR products)1Z0651.25Z027℗1.2550.50.2515.7525
2AVP1-2nd (Plasmid)1Z0651.25Z027℗1.2550.50.2515.7525
3GFP (PCR2)1Z026℗1.25Z027℗1.2550.50.2515.7525
4RFP (PCR5)1Z026℗1.25Z027℗1.2550.50.2515.7525
0831PCR

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

8/31

OE-PCR

Shimazoe
sampletemplatevolume (ul)10uM Z026℗ (ul)10uM Z027℗ (ul)Z026-Z039 (ul)Z040-Z027 (ul)KOD one (ul)DW (ul)Total (ul)
ASseNHX110.30.31151.410
BGFP10.30.31151.410
CRFP10.30.31151.410
0831OE-PCR2

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

9/1

Ligation

Shimazoe
Number1234
vectorpRS 426pRS 316pRS 423pRS 313
insertAtHKT1AtHKT1AtNHXS1AtNHXS1

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
templatevolume (ul)10uM Z026℗ (ul)10uM Z027℗ (ul)Z026-Z039 (ul)Z040-Z027 (ul)Q5 (ul)DW (ul)Total (ul)
SseNHX151.51.55525750

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

9/1

OE-PCR

Shimazoe
templatevolume (ul)10uM Z026℗ (ul)10uM Z027℗ (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
AVP1-1st11.251.2550.50.2514.7525
AVP1-2nd11.251.2550.50.2514.7525
0901OE-PCR2

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

9/1

Colony PCR

Shimazoe
MaterialVolume
2× Emerald amp MAX PCR Master Mix375
10uM m13f7.5
10uM m13r7.5
DW360
Total750
0901ColonyPCR1
0901ColonyPCR2

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

9/2

Ligation

Tong
MaterialVolume(ul)
SseNHX1 (template)2
Vector (313, 423)2
Ligation High4
Total8

9/2

Transformation

Tong
Ligation products(ul)Competent Cells/(ul)Total(ul)
8 (SseNHX1)2533

on ice 20min

9/2

PCR

Shimazoe
MaterialVolume
DW6.8
KODone10
Z0270.6
Z0650.6
template2
Total20

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

9/2

PCR

Shimazoe
MaterialVolume
5× Q5 Reaction Buffer5
10uM dNTPs0.5
10uM primer①1.25
10uM primer②1.25
template1
Q5 DNA Polymerase0.25
DW15.75
Total25

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

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

9/2

OE-PCR

Shimazoe
MaterialVolume
2× Emerald Amp MAX12.25
10uM Z026℗0.25
10uM Z027℗0.25
AVP1-1st1
AVP1-2nd1
DW10.25
Total25

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

9/2

OE-PCR

Shimazoe
MaterialVolume
5× Q5 Reaction Buffer10
10uM Z026℗2.5
10uM Z027℗2.5
10uM dNTPs1
template⓪5
template①5
template②5
Q5 DNA Polymerase0.5
DW18.5
Total50

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
samplevolume(ul)pRS316(ul)Ligation High(ul)Total(ul)
Mangrin1236
ZrFPS1-1st1236
ZrFPS1-2nd1236
ZrGPD11236
McHKT21236
SdrG1236
SdrG-Fgβ1236

18℃overnight

9/4

Transformation

Ligation products(ul)Competent Cells/(ul)Total(ul)
65561

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

9/4

PCR

Tong
Materialvolume (ul)
DW22.8
Template (pIEG-SSS)1.6
Primer F (Z067)2
Primer R (Z066)2
DNA Pol (Q5)0.4
Reaction buffer8
dNTP mix3.2
Total40

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

9/4

OE-PCR

Shimazoe
MaterialVolume
5× Q5 Reaction Buffer10
10uM Z026℗2.5
10uM Z027℗2.5
10uM dNTPs1
template⓪5
template①5
template②5
Q5 DNA Polymerase0.5
DW18.5
Total50

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
MaterialVolume
5× Q5 Reaction Buffer10
10uM Z0262.5
10uM Z0272.5
10uM dNTPs1
template⓪5
template①5
template②5
Q5 DNA Polymerase0.5
DW18.5
Total50

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
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix40
10uM m13f0.8
10uM m13r0.8
DW38.4
Total80

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

9/4

Ligation

Tong
numbersamplevolume(ul)vectorvolume(ul)Ligation High(ul)Total(ul)
1AtHKT11316,426236
2AtNHXS11423,313236
3SseNHX11423,313236

9/5

PCR

Tong
Materialvolume (ul)
DW22
Template (pIEG-SSS)1
Primer F (Z067)1
Primer R (Z066)1
PrimeSTAR Max25
Total50

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

9/5

OE-PCR

Shimazoe
MaterialVolume
5× Q5 Reaction Buffer12
10uM Z026℗3
10uM Z027℗3
10uM dNTPs1.2
template⓪6
template①6
template②6
Q5 DNA Polymerase0.6
DW22.2
Total60

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
123456
sampleAtHKT1AtHKT1AtNHXS1AtNHXS1SseNHX1SseNHX1
vector426316423313423313
competent cell (ul)203020302030

9/5

PCR

Shimazoe
templatevolume (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)
AVP1-1st1Z0261.25Z0711.2550.50.2515.7525
AVP1-2nd1Z0271.25Z0721.2550.50.2515.7525
AVP1-1st1Z0261.25Z0701.2550.50.2515.7525

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

9/5

OE-PCR

Shimazoe
MaterialVolume
5× Q5 Reaction Buffer10
10uM Z026℗2.5
10uM Z027℗2.5
AVP1-1st2
AVP1-2nd2
10uM dNTPs1
Q5 DNA Polymerase0.5
DW24.5
Total50

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
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix180
10uM m13f7.2
10uM m13r7.2
DW180
Total373.4

0905ColonyPCR1
0905ColonyPCR2
0905ColonyPCR3
0905ColonyPCR4

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

9/5

Genome extracion

Tamukai
Materialvolume (ul)
Proteinase K1.2ul
Extraction Reagent1.2ml

・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
MaterialVolume(ul)
2× Gflex PCR Buffer25
10uM Z026℗1
10uM Z027℗1
template⓪1
template①1
template②1
Tks Gflex DNA Polymerase1
DW19
Total50

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

9/5

Ligation

Shimazoe
insertvolume(ul)vectorvolume(ul)Ligation High(ul)Total(ul)
AtHKT12316248
AtHKT12426248
AtNHXS12423248
AtNHXS12313248
SseNHX12423248
SseNHX12313248

9/6

Transformation

Shimazoe
Ligation products(ul)Competent Cells/(ul)Total(ul)
83038

9/6

Colony PCR

Shimazoe
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix125
10uM m13f2.5
10uM m13r2.5
DW120
Total250

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

9/6

OE-PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer25
10uM Z026℗1
10uM Z027℗1
AVP1-1st1
AVP1-2nd 1
Tks Gflex DNA Polymerase1
DW20
Total50

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

9/6

PCR

Shimazoe
MaterialVolume
5× Q5 Reaction Buffer5
10uM primer①1.25
10uM primer②1.25
10uM dNTPs0.5
template1
Q5 DNA Polymerase0.25
DW15.75
Total25

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
plasmidplasmid(ul)10×K buffer(ul)10×H buffer(ul)10× BSA(ul)EcoRⅠ(ul)NcoⅠ(ul)PstⅠ(ul)DpnⅠ(ul)DW(ul)Total/(ul)
38104×422××2840
83104×4×22×2840
pSB1C320×4×2×221040
YCplac1112×4×2×2×3040

9/8

Restriction Enzyme Digestion

Tong
volume(ul)10×H buffer(ul)EcoRⅠ(ul)PstⅠ(ul)DW(ul)Total/(ul)
DNA 40454222740

9/8

Ligation

Tong
samplevolume(ul)vectorvolume(ul)Ligation High(ul)Total(ul)
AtHKT12316248
AtHKT12426248
AtNHXS12423248
AtNHXS12313248
SseNHX12423248
SseNHX12313248

9/8

OE-PCR

Tong
templatevolume (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 ORF52.52.5101550.518.550

0908OE-PCR1

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

9/8

Transformation

Tong
Ligation products(ul)Competent Cells/(ul)Total(ul)
83038

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

9/8

OE-PCR

Tong
template①volume (ul)template②volume (ul)10uM Z026℗ (ul)10uM Z027℗ (ul)2× Prime Star (ul)DW (ul)Total (ul)
AVP1-1st0.5AVP1-2nd0.51110720

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

9/8

PCR

Tong
Materialvolume (ul)
DW22
Template (pIEG-SSS)1
Primer F (Z067)1
Primer R (Z066)1
PrimeSTAR Max25
Total50

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)204221240
YCplac11124223040

9/8

Ligation

Tong
materialvolume(ul)
AVP1-1st1.5
AVP1-2nd1.5
vector1.0
Ligation High4.5
Total9.0

9/9

OE-PCR

Tong
templatevolume (ul)Stalk (ul)Z044 (ul)Z047 (ul)2× Prime Star (ul)DW (ul)Total (ul)
SdrG, Fgβ1111201640

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

9/9

OE-PCR

Tong
templatevolume (ul)10uM Z026℗ (ul)10uM Z027℗ (ul)promoter(ul)terminator(ul)2× Prime Star (ul)DW (ul)Total (ul)
ZrGPD10.5110.50.5106.520

9/9

OE-PCR

Tong
template①volume (ul)template②volume (ul)10uM Z026℗ (ul)10uM Z027℗ (ul)2× Prime Star (ul)DW (ul)Total (ul)
AVP1-1st0.5AVP1-2nd0.50.50.510820

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

9/9

Ligation

Tong
Materialvolume (ul)
AVP1-1st3
YCplac1113
Ligation high6
Total12

9/10

Transformation

Tong
Ligation products(ul)Competent Cells/(ul)Total(ul)
123648

on ice 20min

9/10

Colony PCR

Tong
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix70
10uM m13f1.4
10uM m13r1.4
DW67.2
Total140

0910ColonyPCR1-1
0910ColonyPCR1-2

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

9/10

Colony PCR

Tamukai
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix400
10uM m13f8
10uM m13r8
DW384
Total800

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

9/10

OE-PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer25
10uM Z026℗1
10uM Z027℗1
template⓪1
template①1
template②1
Tks Gflex DNA Polymerase1
DW19
Total50

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

9/10

Colony PCR

Nambu
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix35
10uM m13f0.6
10uM m13r0.6
DW33.6
Total70

0910ColonyPCR3

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

9/10

Restriction Enzyme Digestion

Shimazoe
materialvolume(μl)
EcoT14Ⅰ25
10×H buffer50
λDNA71
DW354
Total500

9/11

Restriction Enzyme Digestion

Nambu
materialvolume(μl)
Mangrin(pRS425)10
BSA4
10×H buffer(10xH)4
Not12
TritonX-1004
DW16
Total40

9/11

Restriction Enzyme Digestion

Nambu
materialvolume(μl)
AVP15
BSA2
10×H buffer(10xH)2
EcoR11
Nco11
Pst11
DW8
Total20

9/11

OE-PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer30
10uM Z026℗1.2
10uM Z027℗1.2
template⓪1.2
template①1.2
template②1.2
Tks Gflex DNA Polymerase1.2
DW22.8
Total60

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

9/11

Ligation

Shimazoe
insertvolume(ul)vectorvolume(ul)Ligation High(ul)Total(ul)
GFP1426124
GFP1316124
RFP1426124
RFP1316124

9/11

PCR

Tamukai
MaterialVolume(ul)
2×Emerald Amp buffer25
Z0030.5
Z0040.5
DW24
Total50

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

9/11

PCR

Tamukai
materialvolume (ul)
DW47.8
5× Q5 Reaction Buffer16
10uM dNTPs 6.4
Q5 DNA Polymerase 0.8
10uM Z0054
10uM Z0284
template(pFA6a-hphMX)1
Total80

ENA1Δ1,2,5

9/11

PCR

Tamukai
materialvolume (ul)
DW45.8
5× Q5 Reaction Buffer16
10uM dNTPs 6.4
Q5 DNA Polymerase 0.8
10uM Z0054
10uM Z0284
template(yeast genome)1
Total80

NHA1

9/11

Ligation

Shimazoe
insertvolume(ul)vectorvolume(ul)Ligation High(ul)Total(ul)
AtHKT11316124
AtHKT11426124
AtNHXS11423124
AtNHXS11313124
SseNHX11423124
SseNHX11313124

9/12

Restriction Enzyme Digestion

Nambu
materialvolume(μl)
CIAP1
pRS426 or pRS4252
BSA4
10×H buffer(10xH)4
Not12
TritonX-1004
DW23
Total40


materialvolume(μl)
AVP1(on pSB1C3)5
BSA2
10×H buffer(10xH)2
Not11
TritonX-1004
DW8
Total20

materialvolume(μl)
AVP1(on pSB1C3)5
BSA2
10×H buffer2
Nco11
EcoR11
Pst11
DW8
Total20

4sample(3,6,7,11)

9/12

Ligation

Shimazoe
MaterialVolume(ul)
Ligation high4
pRS425 Not1 CIAP2
AVP1 Not1 ③2
Total8

9/13

Transformation

Shimazoe
Ligation products(ul)Competent Cells/(ul)Total(ul)
83038

9/13

OE-PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer30
10uM Z017℗1.2
10uM Z018℗1.2
template⓪6
template①6
template②6
Tks Gflex DNA Polymerase1.2
DW8.4
Total60

0913OE-PCR1

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

9/13

OE-PCR

Shimazoe
MaterialVolume(ul)
2× Prime Star Max5
Z0471
Z074 or Z0481
template1
DW2
Total10

98℃* 10s
55℃* 15s
68℃* 10s
(*× 35 cycle)

9/13

PCR

Shimazoe
materialvolume (ul)
2× Prime Star Max5
10uM Z0471
10uM Z0741
template0.5
DW2.5
Total10

9/13

Colony PCR

Shimazoe
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix10
10uM m13f0.2
10uM m13r0.2
DW9.6
Total20

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

9/13

OE-PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer5
10uM Z0440.2
10uM Z0470.2
template①0.5
template②0.5
Tks Gflex DNA Polymerase0.1
DW3.5
Total10

Stalk-SdrG, Stalk-FgβF3
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 2min
68℃ 2min
4℃ ∞
(*× 30 cycle)

9/14

Ligation

Nambu
MaterialVolume(ul)
Ligation high3
pRS4251
AVP1(3,7)2
Total6

9/14

Restriction Enzyme Digestion

Nambu
materialvolume(μl)
sample(404/910/938)10/5/3
EcoR12
10×H buffer4
Pst12
DW22/27/32
Total40

9/14

Colony PCR

Shimazoe
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix62.5
10uM m13f1.25
10uM m13r1.25
DW60
Total125

0914ColonyPCR

SseNHX1/AVP1
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 2min
72℃ 2min
4℃ ∞
(*× 30cycle)

9/14

Restriction Enzyme Digestion

Nambu
sample404910938
volume(ul)1053
10×H buffer(10xH)444
EcoR1222
Not1222
DW222732
Total404043

0914restriction

9/15

OE-PCR

Tong
MaterialVolume(ul)
2× Gflex PCR Buffer30
10uM Z017℗1.2
10uM Z018℗1.2
template pro6
template term6
template ORF6
Tks Gflex DNA Polymerase1.2
DW8.4
Total60

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

9/15

OE-PCR

Tong
MaterialVolume(ul)
2× Prime Star Max20
Z0474
Z0744
template4
DW8
Total40

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

9/15

OE-PCR

Tong
numbergene(plasmid)strainmarker
1×(pRS426)WTURA3
2AtHKT1(pRS426)WTURA3
3Mangrin(pRS316)G19URA3
4×(pRS316)G19URA3
5×(pRS316)WTURA3
6Mangrin(pRS316)WTURA3

9/15

OE-PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer25
10uM Z017℗1
10uM Z0431
template①3
template②3
Tks Gflex DNA Polymerase1
DW16
Total50

ZrGPD1
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)

9/15

Restriction Enzyme Digestion

Nambu
materialvolume(μl)
ZrFPS1-1st7
EcoR10.5
Nco10.5
10×K buffer 1
BSA1
Total10


materialvolume(μl)
ZrFPS1-2nd (718)4
Spe12
Nco12
10×K buffer 4
BSA4
DW24
Total40


materialvolume(μl)
pRS426 2
EcoR12
Spe12
10×H buffer4
CIAP1
DW29
Total40


materialvolume(μl)
pRS316 2
EcoR12
Spe12
10×H buffer4
CIAP1
DW29
Total40

0915RestrictionEnzymeDigestion

9/16

PCR

Tong
materialvolume (ul)
2× Prime Star Max20
10uM Z0474
10uM Z0744
template2
DW10
Total40

Stalk
98℃* 10s
55℃* 15s
72℃* 10s
4℃ ∞
(*× 30 cycle)

9/16

PCR

Tong
MaterialVolume(ul)
2× Gflex PCR Buffer30
10uM Z017℗1.2
10uM Z018℗1.2
template⓪6
template①6
template②6
Tks Gflex DNA Polymerase1.2
DW8.4
Total60

ZrGPD1
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 35 cycle)

9/16

PCR

Tong
materialvolume (ul)
2× Prime Star Max5
10uM Z0471
10uM Z0741
template0.5
DW2.5
Total10

Stalk

9/16

PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer25
10uM primer①1
10uM primer②1
template1
Tks Gflex DNA Polymerase1
DW21
Total50

0916PCR4

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

9/16

OE-PCR

Tong
materialvolume (ul)
2× Prime Star Max20
10uM Z017℗1
10uM Z018℗1
promoter3
SseNHX13
terminator3
DW9
Total40

9/16

Restriction Enzyme Digestion

Nambu
sample1203(RFP)1204(GFP)
volume(ul)21
10×H buffer(10xH)22
EcoR111
Not111
DW1415
Total2020

9/17

OE-PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer30
10uM Z017℗1.2
10uM Z018℗1.2
template⓪6
template①6
template②6
Tks Gflex DNA Polymerase1.2
DW8.4
Total60

0917OE-PCR

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

9/17

PCR

Shimazoe
materialvolume (ul)
5× Q5 Reaction Buffer5
10uM dNTPs 0.5
Q5 DNA Polymerase 0.25
DW15.75
10uM primer①1.25
10uM primer②1.25
template1
Total25

pro/term/Sse
Z017,Z039/Z040,Z018/Z041,Z042 (primer)
P079/P079/317 (template)
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)

9/17

PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer12.5
10uM primer①1
10uM primer②1
template1
Tks Gflex DNA Polymerase1
DW8.5
Total25

0917PCR2

pro,term,Sse ORF
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 30 cycle)

9/17

Transformation

Nambu
volume(ul)Competent Cells(ul)Total(ul)
DNA J0445011011

volume(ul)Competent Cells(ul)Total(ul)
DNA 0.51010.5

DNA J04450…2017 Kit Plate 1 24A

9/18

OE-PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer30
10uM Z017℗1.2
10uM Z018℗1.2
template⓪3
template①3
template②3
Tks Gflex DNA Polymerase1.2
DW17.4
Total60

ZrGPD1,SseNHX1
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 30 cycle)

9/18

Ligation

Tong
MaterialVolume(ul)
Ligation high2
pRS4251
AVP1(3)1
Total4

MaterialVolume(ul)
Ligation high3.5
pRS4250.5
AVP1(3)3
Total7

9/18

Inverse PCR

Shimazoe
materialvolume (ul)
2× Prime Star Max25
10uM Z0391
10uM Z0401
template(938)0.5
DW22.5
Total50

98℃* 10s
55℃* 5s
72℃* 40s
(*× 30 cycle)

9/18

SLiCE

Shimazoe
MaterialVolume(ul)
vector0.5
insert1
10× SLiCE Buffer1
SLiCE 1
DW6.5
Total10

SseNHX1/GPD1


MaterialVolume(ul)
vector0.5
insert①1
insert②1
10× SLiCE Buffer1
SLiCE 1
DW5.5
Total10

0918SLiCE

Fgβ/SdrG

9/18

Transformation

Shimazoe
Ligation productsvolume(ul)Competent Cells/(ul)Total(ul)
SseNHX1/GPD1105060
Fgβ/SdrG105060

9/19

OE-PCR

Tong
MaterialVolume(ul)
2× Gflex buffer12.5
Z0440.5
Z0470.5
SdrG or Fgβ1
Stalk1
Gflex DNA polymerase0.5
DW9
Total25

9/19

Colony PCR

Tong
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix82.5
10uM m13f1.65
10uM m13r1.65
DW79.2
Total165

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

9/19

Inverse PCR

Tong
materialvolume (ul)
2× Prime Star Max25
10uM Z0391
10uM Z0401
template(938)0.6
DW22.5
Total50

98℃* 10s
55℃* 5s
72℃* 40s
(*× 30cycle)

9/19

PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer25
10uM primer①1
10uM primer②1
template1
Tks Gflex DNA Polymerase1
DW21
Total50

template(SseNHX1/ZrGPD1/Stalk)
primer(Z041,Z042/Z041,Z042/Z047,Z074)

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

9/19

Colony PCR

Tong
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix17.5
10uM Z0470.35
10uM Z0740.35
DW16.8
Total35


98℃ 30s
98℃* 10s
60℃* 30s
72℃* 3min
72℃ 2min
4℃ ∞
(*× 30cycle)

9/19

Restriction Enzyme Digestion

Tong
materialvolume (ul)
PCR products 42
Dpn12
Total44

0919Dpn1

37℃/1h

9/20

Inverse PCR

Tong
materialvolume (ul)
2× Prime Star Max25
10uM Z0391
10uM Z0401
template(938) 1/2000.6
DW22.4
Total50

0920InversePCR

98℃* 10s
55℃* 5s
72℃* 40s
(*× 30cycle)

9/20

Restriction Enzyme Digestion

Tong

Dpn1 digestion

9/20

PCR

Tong
materialvolume (ul)
2× Prime Star Max25
10uM Z0471
10uM Z0741
template①/②1/1
DW22
Total50

98℃* 10s
55℃* 15s
72℃* 10s
4℃ ∞
(*× 30cycle)

9/20

Ligation

Shimazoe
MaterialVolume(ul)
Ligation high2
vector 1
insert1
Total4

Mangrin : pRS426
AVP1 : pRS425

9/20

Nanodrop

Nambu
1234
222.6 ng/ul181.2 ng/ul232.1 ng/ul128.7 ng/ul

9/20

Restriction Enzyme Digestion

Nambu
sample1234
volume(ul)6669
10×H buffer(10xH)3333
EcoR11.51.51.51.5
Pst11.51.51.51.5
DW18181815
Total30303030

0920restriction

put in incubator at 37℃

9/21

Qubit^tm dsDNA HS Assay Kit

Shimazoe
MaterialVolume(ul)
dsDNA buffer200
dsDNA reagent1
dsDNA solution1
Total202

9/21

Ligation

Shimazoe
MaterialVolume(ul)
Ligation high6
vector 426/3162/2
ZrFPS1-1st 2
ZrFPS1-2nd 2
Total12

9/21

SLiCE

Shimazoe
MaterialVolume(ul)
DW5.5
pIEG-SSS 1/201
FgβF3 1/101
Stalk 0.5
10× SLiCE Buffer1
SLiCE 1
Total10

MaterialVolume(ul)
DW6.1
pIEG-SSS 1/201
SdrG 0.4
Stalk 0.5
10× SLiCE Buffer1
SLiCE 1
Total10

9/21

PCR

Shimazoe
materialvolume (ul)
2× Prime Star Max12.5
10uM Z0411
10uM Z0421
template 1/2001
DW9.5
Total25

template : 317(SseNHX1), 506(ZrGPD1)
98℃* 10s
55℃* 5s
72℃* 10s
(*× 30cycle)

9/21

PCR

Shimazoe
materialvolume (ul)
2× Prime Star Max25
10uM Z0412
10uM Z0422
template 1/2001
DW20
Total50

template : 317(SseNHX1), 506(ZrGPD1)
98℃* 10s
55℃* 5s
72℃* 10s
(*× 30cycle)

9/21

Transformation

Shimazoe
MaterialVolume(ul)
SLiCE products(①~④)4
competent cell50
Total54

template(SseNHX1/ZrGPD1/Stalk)
on ice 30min
42℃45s
on ice 2min
SOC 500ul
37℃ 40min

9/21

Transformation

Shimazoe
MaterialVolume(ul)
In-Fusion products(⑤,⑥)4
competent cell50
Total54

9/21

Transformation

Tong
yeastplasmid
WT(547)・AVP1(YCplac111)
・AtNHXS1(pRS423)(938)
・empty(pRS316)
WT・AVP1(YCplac111)
・AtNHXS1(pRS313)(938)
・empty(pRS316)
WT・AVP1(YCplac111)
・empty(pRS313)
・empty(pRS316)
WT・AtNHXS1(pRS423)(938)
・Mangrin(pRS316)
WT・empty(YCplac111)
・AtNHXS1(pRS313)
・empty(pRS316)
ΔENA1(559)・AVP1(YCplac111)
・AtNHXS1(pRS423)
・empty(pRS316)

competent cell : 10ul
plasmid : 0.5ul
Solution3 : 100ul
vortex 30s
37℃/35min

9/22

SLiCE

Shimazoe
MaterialVolume(ul)
DW5.5
pIEG-SSS 1/201
FgβF3 1/101
Stalk 0.5
10× SLiCE Buffer1
SLiCE 1
Total10

MaterialVolume(ul)
DW5
pIEG-SSS 1/201
SdrG 1/41
Stalk 1/21
10× SLiCE Buffer1
SLiCE 1
Total10

9/22

In Fusion

Shimazoe
MaterialVolume(ul)
DW5
pIEG-SSS 1/201
FgβF3 1/101
Stalk 0.5
5× In-Fusion HD Enzyme Premix2
Total10

MaterialVolume(ul)
DW5
pIEG-SSS 1/201
SdrG 1/41
Stalk 1/21
5× In-Fusion HD Enzyme Premix2
Total10

9/22

Colony PCR

Shimazoe
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix25
10uM m13f0.5
10uM m13r0.5
DW24
Total50

MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix15
10uM Z0440.3
10uM Z0470.3
DW14.4
Total30

0922ColonyPCR


98℃ 30s
98℃* 10s
60℃* 30s
72℃* 3min
72℃ 2min
4℃ ∞
(*× 30cycle)

9/22

PCR

Tong
MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
10uM Z0051
10uM Z0281
template1
enzyme 0.4
DW25.4
Total40

Cassette for ΔENA1,2,5
98℃* 10s
55℃* 15s
72℃* 2min
(*× 30 cycle)

9/22

Spot

Tong
Number Material< /th>
547 WT
554 ΔNHA1
555 G19
557 ΔNHA1,ΔENA1
559 ΔENA1
ΔENA1.2.5, ΔNHA1
564 Z.roxii

Cassette for ΔENA1,2,5
98℃* 10s
55℃* 15s
72℃* 2min
(*× 30 cycle)

9/22

Restriction Enzyme Digestion

Nambu
sample1087(AtNHXS1)910(AtHKT1)
volume(ul)2020
10×H buffer(10xH)44
EcoR111
Not111
DW1414
Total4040

enzyme inactivation 60℃ 15min

9/22

Restriction Enzyme Digestion

Nambu
samplepSB1C3
volume(ul)20
10×H buffer(10xH)4
EcoR12
Not12
Dpu12
DW10
Total40

enzyme inactivation 80℃ 20min

9/22

Transformation

Nambu
volume(ul)Competent Cells(ul)Total(ul)
Mangrin 40421012

9/23

Colony PCR

Shimazoe
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix35
10uM m13f0.7
10uM m13r0.7
DW33.6
Total70

MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix85
10uM Z0441.7
10uM Z0471.7
DW81.6
Total170

0923ColonyPCR


98℃ 30s
98℃* 10s
60℃* 30s
72℃* 3min
72℃ 2min
4℃ ∞
(*× 30cycle)

9/23

PCR

Tong
MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
10uM Z0031
10uM Z0041
Yeast Genome1
enzyme 0.4
DW25.4
Total40

Cassette for ΔENA1,2,5
98℃* 10s
55℃* 15s
72℃* 4min
(*× 35 cycle)

9/23

Restriction Enzyme Digestion

Tong
materialvolume (ul)
Plasmid(404,716,910,3)10
10×H buffer2
BSA2
Triton X-1002
Not11
DW3
Total20

404,716: Mangrin(pRS716)
910: AtHKT1(pRS426)
3: AVP1(pSB1C3)

9/23

Colony PCR

Nambu
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix18.75
10uM m13f0.375
10uM m13r0.375
DW18
Total37.5

0923colonyPCR101

98℃ 30s
98℃* 10s
60℃* 30s
72℃* 3min
72℃ 2min
4℃ ∞
(*× 30cycle)

9/24

Colony PCR

Shimazoe
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix120
10uM Z0442.4
10uM Z0472.4
DW115.2
Total240

0924ColonyPCR

9/24

Ligation

Shimazoe
MaterialVolume(ul)
Ligation high4
vector 426/3161/1
McHKT23
Total8

9/24

Ligation

Nambu
numberinsertvolume(ul)vectorvolume(ul)Ligation High(ul)Total(ul)
1.2.1910(heat)1.51.20.524
1.2.2910(column)1.51.20.524
1.2.31087(heat)1.51.20.524
1.2.41087(column)1.51.20.524
PL1910(heat)1.5pSB1C30.524
PL2910(column)1.5pSB1C30.524
PL31087(heat)1.5pSB1C30.524
PL41087(column)1.5pSB1C30.524

9/24

Transformation

Nambu
volume(ul)Competent Cells(ul)Total(ul)
DNA 21012

DNA(1.2.1/1.2.2/1.2.3/1.2.4/PL1/PL2/PL3/PL4)

9/25

Ligation

Shimazoe
MaterialVolume(ul)
Ligation high2
insert1
vector1
Total4

insert4047161304130613101118130113021303
vector426426313313313425313313313

9/25

Colony PCR

Nambu
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix75
10uM m13f1.5
10uM m13r1.5
DW72
Total150

AtNHXS1(pSB1C3)/AtHKT1(pRS426)/McHKT2(pRS426,pRS316)

98℃ 30s
98℃* 10s
60℃* 30s
72℃* 3min
72℃ 2min
4℃ ∞
(*× 30cycle)

9/25

Restriction Enzyme Digestion

Nambu
sampleDNA(pSB1C3 Linear)
volume(ul)5
10×M buffer2
Xba11
Pst11
Dpn11
DW10
Total20

enzyme inactivation 80℃ 20min

9/25

Restriction Enzyme Digestion

Nambu
sample130113021303130413061310
volume(ul)10101010102
10×H buffer222222
EcoR1111111
Pst1111111
DW6666614
Total202020202020

enzyme inactivation 60℃ 15min

9/26

Colony PCR

Shimazoe
MaterialVolume
5× Q5 Reaction Buffer36
10uM dNTPs3.6
10uM m13r9
10uM m13f9
Q5 DNA Polymerase1.8
DW120.5
Total180


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

9/26

PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer12.5
10uM primer①0.5
10uM primer②0.5
template0.5
Tks Gflex DNA Polymerase0.5
DW10.5
Total25

ZrFPS1-1stZrFPS1-2nd
primerZ041, Z043Z077, Z042
template927718

0926PCR

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

9/26

Restriction Enzyme Digestion

Nambu
samplePlasmid(AVP1.pSB1C3)
volume(ul)20
10×H buffer4
EcoR11
Pst11
CIAP1
DW13
Total40

9/26

Restriction Enzyme Digestion

Nambu
sampleλDNA
volume(ul)71
10×H buffer50
Sty13
DW376
Total500

9/27

Transformation(yeast)

Tong
yeastplasmid
34WT ・empty(pRS425)
・empty(pRS316)
・Mangrin(pRS316)
35WT ・AVP1(YCplac111)
・SseNHX1(pRS423)
・empty(pRS316)
36WT ・AVP1(YCplac111)
・AtNHXS1 (pRS423)
・Mangrin(pRS316)
37WT ・AVP1(YCplac111)
・AtNHXS1(pRS313)
・Mangrin(pRS316)
38WT ・AVP1(YCplac111)
・(1301) SseNHX1(pRS423)
・Mangrin(pRS316)
39WT ・empty(pRS425)
・(1432)SdrG(pIEG-SSS:HIS)
・(1204)GFP(pRS316)
40WT ・empty(pRS425)
・(1444)SdrG
・(1204)GFP
41WT ・empty(pRS425)
・(1448)SdrG
・(1204)GFP(pRS316)

9/28

OD measure

Tong
numberODnumberOD
74.8206.6
85.8216.2
96.4225.9
106.823×
116.624×
126.025×
136.626×
146.427×
15×284.1
166.6294.7
176.9303.8
186.631/RFP
196.432/GFP

9/28

PCR

Shimazoe
materialvolume (ul)
2× Prime Star Max12.5
10uM primer①1
10uM primer②1
template0.5
DW10
Total25

FgβSdrGstalk for Fgβstalk for SdrG
template342, 1/300752, 1/100gBlockgBlock
primerZ086
Z084
Z086
Z045
Z046
Z087
Z074
Z087

9/28

Inverse PCR

Shimazoe
materialvolume (ul)
2× Prime Star Max25
10uM Z0852
10uM Z0882
template(pIEG-SSS)1
DW20
Total50

98℃* 10s
55℃* 5s
72℃* 40s
(*× 30 cycle)

9/28

OE-PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer25
10uM Z0411
10uM Z0421
ZrFPS1-1st1
ZrFPS1-2nd1
Tks Gflex DNA Polymerase1
DW20
Total50

0928OE-PCR

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

9/28

PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer25
10uM primer①1
10uM primer②1
template1
Tks Gflex DNA Polymerase1
DW21
Total50

0928PCR2

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

9/28

Restriction Enzyme Digestion

Nambu
sampleMangrinAVP1・pSB1C3
volume(ul)2020
10×H buffer44
EcoR111
Pst111
CIAP×1
DW1413
Total4040

0928restriction1

9/28

PCR

Nambu
StalkFgβF3SdrGAtHKT1(910)
2× Prime Star Max (ul)20202020
F.primer 1(Z082)1(Z080)1(Z078)1(Z094)
R.primer 1(Z083)1(Z081)1(Z079)1(Z095)
template1(gBlock)×0.5(752,1/100)1(910,1/100)
DW171817.517
Total40404040

0928PCR101

9/28

Restriction Enzyme Digestion

Nambu
sample910-1910-2
volume(ul)2020
10×M buffer44
Xba111
Pst111
DW1414
Total4040

9/29

Restriction Enzyme Digestion

Tong
materialvolume (ul)
plasmid(716/1301/1304/938)10/15/15/15
10×H buffer4
EcoR10.5
Pst10.5
DW25/20/20/20
Total40

insert


materialvolume (ul)
plasmid(6)20
SAP 1
10×H buffer8
EcoR11
Pst11
DW49
Total80

Backbone


materialvolume (ul)
plasmid(910)15
10×M buffer4
Xba10.5
Pst10.5
DW20
Total40

insert


materialvolume (ul)
plasmid(6)10
SAP 0.5
10×M buffer4
Xba10.5
Pst10.5
DW24.5
Total40

Backbone

9/29

PCR

Tong
MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
10uM primer①1
10uM primer②1
template 1
enzyme 0.4
DW25.4
Total40

SdrGFgβStalk for FgβStalk for SdrG
template 752, 1/100342, 1/300gBlockgBlock
primerZ045, Z086Z084, Z086Z046, Z087Z074, Z087

9/29

OD measure

Tong
numberODnumberODnumberOD
7-A1.1311-A1.5916-A0.54
7-B1.1211-B1.5716-B0.58
8-A1.2412-A0.8117-A1.49
8-B1.1912-B0.8017-B1.55
9-A1.1113-A1.4820-A0.87
9-B1.1613-B1.4420-B0.72
10-A1.5914-A1.5328-A1.13
10-B1.6114-B1.5228-B1.15

9/29

PCR

Shimazoe
MaterialVolume(ul)
2× Gflex PCR Buffer20
10uM primer①1
10uM primer②1
template1
Tks Gflex DNA Polymerase1
DW16
Total40

FgβSdrG
template342752
primerZ089, Z090Z091, Z092

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

9/29

PCR

Shimazoe
materialvolume (ul)
2× Prime Star Max12.5
10uM primer①1
10uM primer②1
template0.5
DW10
Total25

Stalk
for Fgβ
Stalk
for SdrG
Fgβ
for OE-PCR
SdrG
for OE-PCR
templategBlockgBlock342gBlock
primerZ086
Z084
Z074
Z087
Z086
Z045
Z093
Z087

0929PCR1

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

9/29

SLiCE

Shimazoe
MaterialVolume(ul)
DW7
pRS4230.5
ZrFPS1-OE0.5
10× SLiCE Buffer1
SLiCE 1
Total10

9/29

Restriction Enzyme Digestion

Nambu
sampleSdrGFgβ
volume(ul)3434
10×H buffer44
EcoR111
Pst111
Total4040

37℃ 3h

9/29

PCR

Nambu
MaterialVolume(ul)
2× Prime Star Max (ul)12.5
F.primer (Z082) 1
R.primer (Z083) 1
template (gBlock)1
DW9.5
Total25

0929PCR101

98℃* 10s
55℃* 5s
72℃* 11s
(*× 30cycle)

9/29

Ligation

Nambu
insertvolume(ul)vectorvolume(ul)Ligation High(ul)Total(ul)
Mangrin 2AVP1・pSB1C3(E,P cut)12.55.5
ZrGPD1(1304) 2 AVP1・pSB1C3(E,P cut)12.55.5
SseNHX1(1302)2 AVP1・pSB1C3(E,P cut)12.55.5
Fgβ 2 AVP1・pSB1C3(E,P cut)12.55.5
SdrG2 AVP1・pSB1C3(E,P cut)12.55.5
AtNHXS1(1087)2 AVP1・pSB1C3(E,P cut)12.55.5
Control(MilliQ)2 AVP1・pSB1C3(E,P cut)12.55.5

9/29

Transformation

Nambu
volume(ul)Competent Cells(ul)Total(ul)
Mangrin 21012
ZrGPD1(1304) 21012
SseNHX1(1302)21012
Fgβ21012
SdrG21012
AtNHXS1(1087)21012
Control(MilliQ)21012

20℃ 40h

9/29

PCR

Tong
MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
10uM primer①1
10uM primer②1
template 1
enzyme 0.4
DW25.4
Total40

FgβStalk for FgβStalk for Fgβ
template 342, 1/300gBlock,1/10gBlock,1/10
primerZ045, Z086Z046, Z087Z093, Z087

SdrGFgβStalk for SdrG
template 752, 1/100342, 1/300gBlock
primerZ045, Z086Z084, Z086Z074, Z087

9/29

Ligation

Tong
insertvolume(ul)vectorvolume(ul)Ligation High(ul)Total(ul)
AVP1・pSB1C3(E,P cut)11301124
AVP1・pSB1C3(E,P cut)11304124
AVP1・pSB1C3(E,P cut)1716124
AVP1・pSB1C3(E,P cut)1938124

insertvolume(ul)vectorvolume(ul)Ligation High(ul)Total(ul)
AVP1・pSB1C3(E,P cut)1910348

9/29

Transformation

Tong
numberplasmidYeast Type
42pRS425
pRS423
(404)Mangrin(pRS316)
WT

9/30

PCR

Tong
MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
10uM primer①1
10uM primer②1
template 1
enzyme 0.4
DW25.4
Total40

number
template 342, 1/300Stalk gBlock 1/10Stalk gBlock 1/1013 gBlock13 gBlock13 1/5013 1/50
primerZ045, Z086Z046, Z087Z093, Z087Z046, Z087Z093, Z087Z046, Z087Z093, Z087

9/30

OE-PCR

Tong
MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
10uM primerF1
10uM primerR1
template 11
template 21
enzyme 0.4
DW24.6
Total40

numberTemplate1Template2Primer
1Z045, Z086Z074, Z087Z086, Z087
2Z084, Z086Z074, Z087Z086, Z087

98℃* 10s
55℃* 5s
72℃* 2m30s
(*× 30cycle)

9/30

OE-PCR

Tong
MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
10uM primer①1
10uM primer②1
template 11
template 21
enzyme 0.4
DW24.4
Total40

template2: ①Z046-Z087, ②Z093-Z087

9/30

Transformation

Tong
volume(ul)Competent Cells(ul)Total(ul)
plasmid(1432/1444)11011

on ice 15min/42℃ 45s/on ice 1min

10/1

Colony PCR

Shimazoe
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix90
10uM m13f1.8
10uM m13r1.8
DW86.4
Total180

10/1

Restriction Enzyme Digestion

Nambu
sampleStalkAVP1・pSB1C3
volume(ul)1910
10×H buffer3×
10×M buffer×2
Xba1×1
EcoR11×
Pst111
DW67
Total3021

10/1

Colony PCR

Nambu
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix162.5
10uM m13f3.25
10uM m13r3.25
DW156
Total325

1001colonyPCR101
1001colonyPCR102

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 3min
72℃ 2min
15℃ ∞
(*× 30cycle)

10/1

Colony PCR

Tong
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix2.5
VF2 0.1
VR0.1
DW2.3
Total5

MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix2.5
VF20.1
Z0270.1
DW2.3
Total5

10/1

PCR

Tong
MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
Z0861
Z0871
template 11
template 21
enzyme 0.4
DW24.4
Total40

number1234
template 1Z045, Z086Z045, Z086Z084, Z086Z084, Z086
template 2Z046, Z087Z093, Z087Z046, Z087Z093, Z087

10/1

In Fusion

Shimazoe
MaterialVolume(ul)
DW5
pIEG-SSS 1/31
Fgβ 1/101
Stalk for Fgβ1/21
5× In-Fusion HD Enzyme Premix2
Total10

MaterialVolume(ul)
DW5
pIEG-SSS 1/31
SdrG 1/41
Stalk for SdrG1
5× In-Fusion HD Enzyme Premix2
Total10

10/1

Colony PCR

Shimazoe
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix160
10uM Z0863.2
10uM Z0873.2
DW153.6
Total320

0922ColonyPCR


98℃ 10s
98℃* 10s
60℃* 30s
72℃* 3min
72℃ 3min
(*× 30cycle)

10/2

Colony PCR

Nambu
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix18.75
10uM m13f0.375
10uM m13r0.375
DW18
Total37.5

1002colonyPCR101

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m45s
72℃ 2min
15℃ ∞
(*× 30cycle)

10/2

Colony PCR

Nambu
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix150
10uM m13f3
10uM m13r3
DW144
Total300

1002colonyPCR102

4(Mangrin)/12(ZrGPD1)/32(AtNHXS1)/33(Fgβ)/44(SdrG)

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 3min
72℃ 2min
15℃ ∞
(*× 30cycle)

10/3

Nanodrop

Nambu
Mangrin(4)ZrGPD1(12)AtNHXS1(32)Fgβ(33)SdrG(44)AtHKT1(910)
267.7 ng/ul211.6 ng/ul243.5 ng/ul222.1 ng/ul250.8 ng/ul23.7 ng/ul

10/2

Colony PCR

Tong
1~14716(Mangrin)pRS31615163,8,14
15~261304(ZrGPD1)pRS423250119,20,22,24,26
27~42938(AtNHXS1)pRS423202430,31,33
43~54,66,711301(SseNHX1)pRS4232957(VR,VF2)/2811(VF2,Z027)40,41,44,47
55~65,67,(68),69,70910(AtHKT1)pRS4232670(VF2,Z027)/2816(VR,VF)55,63,67,68,69,70

1002colonyPCR102

4(Mangrin)/12(ZrGPD1)/32(AtNHXS1)/33(Fgβ)/44(SdrG)

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 3min
72℃ 2min
15℃ ∞
(*× 30cycle)

10/2

PCR

Tong
MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
10uM primerF1
10uM primerR1
template 1
enzyme 0.4
DW25.4
Total40

template 752, 1/100gBlock, 1/10
primerZ078, Z079Z082, Z083

SdrG, Stalk

10/2

PCR

Tong
MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
10uM primerF(Z080)1
10uM primerR(Z081)1
enzyme 0.4
DW26.4
Total40

Fgβ

10/2

Restriction Enzyme Digestion

Tong
sample3/8/14/19/20/22/24/26/30/31/33/40/41/44/47
volume(ul)10
10×H buffer4
EcoR10.5
Pst10.5
DW25
Total40

10/2

Ligation

Shimazoe
MaterialVolume(ul)
Ligation high3
vector 1
insert2
Total6

10/2

PCR

Shimazoe
MaterialVolume
5× Q5 Reaction Buffer4
10uM dNTPs1.6
10uM Z0940.5
10uM Z0390.5
template(p079) 0.5
DW12.7
PrimeSTAR HS0.2
Total20

MaterialVolume
5× Q5 Reaction Buffer4
10uM dNTPs1.6
10uM Z0950.5
10uM Z0770.5
template(718)0.5
DW12.7
PrimeSTAR HS0.2
Total20

Fgβ

10/3

Restriction Enzyme Digestion

Nambu
sampleMangrin(4)ZrGPD1(12)AtNHXS1(32)Fgβ(33)SdrG(44)
volume(ul)55555
10×H buffer22222
EcoR111111
Pst111111
DW1111111111
Total2020202020

sampleAtHKT1AVP1・pSB1C3
volume(ul)3020
10×M buffer43
Xba111
Pst111
CIAP ×1
DW44
Total4030

10/3

Liquid Culture

Tong

21,28,53,57 Amp

10/3

Ligation

Tong
insertvolume(ul)vectorvolume(ul)Ligation High(ul)Total(ul)
gBlock1pRS426(Sma1 cut)/326124

10/3

Ligation

Tong
volume(ul)vectorvolume(ul)Ligation High(ul)Total(ul)
Z082-Z83/DW1pSB1C3124

10/3

Restriction Enzyme Digestion

Tong
samplevolume(ul)
gBlock(McHKT2)20
10×H buffer10
EcoR11
Pst11
DW68
Total100

10/3

PCR

Tong
MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
10uM Z0941
10uM Z0951
template①1
template②1
template③1
DW23.4
PrimeSTAR HS0.4
Total40

10/3

PCR

Shimazoe
MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
10uM Z0801
10uM Z0831
template1
Polymerase0.4
DW25.4
Total40

MaterialVolume
5× Q5 Reaction Buffer8
10uM dNTPs3.2
10uM Z0781
10uM Z0831
template1
Polymerase0.4
DW25.4
Total40

Fgβ-Stalk, SdrG-Stalk

10/4

Nanodrop

Nambu
sample12912172025334046
concentration(ng/ul)182.6199.9323.0246.3264.2283.5192.9187.5212.5199.8

10/4

Restriction Enzyme Digestion

Nambu
sample1/2/9/12/17/20/25/33/40/46
volume(ul)5
10×H buffer2
EcoR11
Pst11
DW11
Total20

10/4

Colony PCR

Nambu
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix42.5
10uM VF20.85
10uM Z0180.85
DW40.8
Total85

98℃ 30s
98℃* 10s
55℃* 30s
72℃* 3min
72℃ 3min
15℃ ∞
(*× 30cycle)

10/4

Liquid Culture

Nambu

AtNHXS1 “2” “3”

10/4

Ligation

Shimazoe
MaterialVolume(ul)
Ligation high2
vector1
insert 1
Total4

C1/LC2/LC3/LC4/LC5/LC2/HC3/H
backbonepSB1C3pRS316pRS316pSB1C3pSB1C3YCplac195YCplac195
insertMcHKT2ZrFPS1ZrFPS1SdrGFgβZrFPS1ZrFPS1

10/4

Restriction Enzyme Digestion

Shimazoe
materialvolume(ul)
DNA15
10×H buffer8
EcoR11
Pst11
DW55
Total80

10/4

Ligation

Shimazoe
MaterialVolume(ul)
Ligation high2
vector1
insert 1
Total4

10/5

Liquid Culture

Nambu

Fgβ 34,35,36,37,38,39

10/5

Colony PCR

Nambu
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix87.5
10uM m13f1.75
10uM m13r1.75
DW84.0
Total175.0

Stalk
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 2m30s
72℃ 3min
15℃ ∞
(*× 30cycle)

10/5

Restriction Enzyme Digestion

Shimazoe
sample volume(ul)
DNA10
10×H buffer2
Sca11
DW7
Total20

RRRRRRRR
FgβFgβFgβFgβSdrGSdrGSdrGSdrG
pIEGpIEG2128pIEGpIEG5357

10/5

Transformation

Tong
LabelingYeast typeGene
43 ΔENA1 empty(pRS425:Leu)
empty(pRS423)
404 Mangrin(pRS326)
44 ΔENA1 empty (pRS425:Leu)
empty (pRS423)
910 AtHKT1(pRS426)
45 ΔENA1 1118 AVP1(YCplac:Leu)
empty (pRS423)
empty (pRS426)
46 ΔENA1 empty (pRS425:Leu)
938 AtNHXS1(pRS423)
empty (pRS426)
47 ΔENA1 empty (pRS425:Leu)
1087 AtNHXS1(pRS313)
empty (pRS426)
48 ΔENA1 empty (pRS425:Leu)
1301 SseNHXS1(pRS423)
empty (pRS426)
49 ΔENA1 empty (pRS425:Leu)
1304 ZrGPD1(pRS423)
empty (pRS426)
50 ΔENA1 empty (pRS425:Leu)
empty (pRS423)
empty (pRS426)
51 564(Zysosaccharomyces rouxii) empty (pRS425:Leu)
empty (pRS423)
404 Mangrin(pRS326)
52 564(Zysosaccharomyces rouxii) empty (pRS425:Leu)
empty (pRS423)
910 AtHKT1(pRS426)
53 564(Zysosaccharomyces rouxii) 1118 AVP1(YCplac:Leu)
empty (pRS423)
empty (pRS426)
54 564(Zysosaccharomyces rouxii) empty (pRS425:Leu)
938 AtNHXS1(pRS423)
empty (pRS426)
55 564(Zysosaccharomyces rouxii) empty (pRS425:Leu)
1087 AtNHXS1(pRS313)
empty (pRS426)
56 564(Zysosaccharomyces rouxii) empty (pRS425:Leu)
1301 SseNHXS1(pRS423)
empty (pRS426)
57 564(Zysosaccharomyces rouxii) empty (pRS425:Leu)
empty (pRS423)
empty (pRS426)






10/5,10/6

Immuno Precipitation

Shimazoe

・SDS-PAGE
・Western Blotting

10/7

Colony PCR

Shimazoe
MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix120
10uM VR2.4
10uM VF2.4
DW115.2
Total240

MaterialVolume(ul)
2× Emerald amp MAX PCR Master Mix100
10uM m13r2
10uM m13f2
DW96
Total200

10/10

Transformation

Tong
LabelingYeast typeURALEUHIS
73 ΔENA1 910AtHKT1(426) AVP1-1(425) 938 AtNHXS1(423)
74 ΔENA1 911 AtHKT1(426) AVP1-1(425) 1301 SseNHX1(423)
75 ΔENA1 empty, pRS426 AVP1-1(425) 938 AtNHXS1(423)
76 ΔENA1 empty, pRS426 AVP1-1(425) 1301 SseNHX1(423)
77 ΔENA1 910 AtHKT1(426) empty, pRS425 938 AtNHXS1(423)
78 ΔENA1 911 AtHKT1(426) empty, pRS425 1301 SseNHX1(423)
79 ΔENA1 910 AtHKT1(426) AVP1-1(425) empty, pRS423
80 ΔENA1 911 AtHKT1(426) AVP1-1(425) empty, pRS423
81 ΔENA1 empty, pRS426 AVP1-1(425) empty, pRS423
82 ΔENA1 empty, pRS426 empty, pRS425 938 AtNHXS1(423)
83 ΔENA1 empty, pRS426 empty, pRS425 1301 SseNHX1(423)
84 ΔENA1 empty, pRS426 empty, pRS425 empty, pRS423
85 ΔENA1,2,5ΔNHA1 910 AtHKT1(426) AVP1-1(425) 938 AtNHXS1(423)
86 ΔENA1,2,5ΔNHA1 911 AtHKT1(426) AVP1-1(425) 1301 SseNHX1(423)
87 ΔENA1,2,5ΔNHA1 empty, pRS426 AVP1-1(425) 938 AtNHXS1(423)
88 ΔENA1,2,5ΔNHA1 empty, pRS426 AVP1-1(425) 1301 SseNHX1(423)
89 ΔENA1,2,5ΔNHA1 910 AtHKT1(426) empty, pRS425 938 AtNHXS1(423)
90 ΔENA1,2,5ΔNHA1 911 AtHKT1(426) empty, pRS425 1301 SseNHX1(423)
91 ΔENA1,2,5ΔNHA1 910 AtHKT1(426) AVP1-1(425) empty, pRS423
92 ΔENA1,2,5ΔNHA1 911 AtHKT1(426) AVP1-1(425) empty, pRS423
93 ΔENA1,2,5ΔNHA1 empty, pRS426 AVP1-1(425) empty, pRS423
94 ΔENA1,2,5ΔNHA1 empty, pRS426 empty, pRS425 938 AtNHXS1(423)
95 ΔENA1,2,5ΔNHA1 empty, pRS426 empty, pRS425 1301 SseNHX1(423)
96 ΔENA1,2,5ΔNHA1 empty, pRS426 empty, pRS425 empty, pRS423
97 ΔENA1,2,5ΔNHA1 NHA1 AVP1-1(425) 938 AtNHXS1(423)
98 ΔENA1,2,5ΔNHA1 NHA1 AVP1-1(425) 1301 SseNHX1(423)
99 ΔENA1,2,5ΔNHA1 NHA1 empty, pRS425 938 AtNHXS1(423)
100 ΔENA1,2,5ΔNHA1 NHA1 empty, pRS425 1301 SseNHX1(423)
101 ΔENA1 910 AtHKT1(426) AVP1(YCplac111) 938 AtNHXS1(423)
102 ΔENA1 911 AtHKT1(426) AVP1(YCplac111) 1301 SseNHX1(423)
103 ΔENA1 empty, pRS426 AVP1(YCplac111) 938 AtNHXS1(423)
104 ΔENA1 empty, pRS426 AVP1(YCplac111) 1301 SseNHX1(423)
105 ΔENA1 910 AtHKT1(426) AVP1(YCplac111) empty, pRS423
106 ΔENA1 911 AtHKT1(426) AVP1(YCplac111) empty, pRS423
107 ΔENA1 empty, pRS426 AVP1(YCplac111) empty, pRS423
108 ΔENA1,2,5ΔNHA1 910 AtHKT1(426) AVP1(YCplac111) 938 AtNHXS1(423)
109 ΔENA1,2,5ΔNHA1 911 AtHKT1(426) AVP1(YCplac111) 1301 SseNHX1(423)
110 ΔENA1,2,5ΔNHA1 empty, pRS426 AVP1(YCplac111) 938 AtNHXS1(423)
111 ΔENA1,2,5ΔNHA1 empty, pRS426 AVP1(YCplac111) 1301 SseNHX1(423)
112 ΔENA1,2,5ΔNHA1 910 AtHKT1(426) AVP1(YCplac111) empty, pRS423
113 ΔENA1,2,5ΔNHA1 911 AtHKT1(426) AVP1(YCplac111) empty, pRS423
114 ΔENA1,2,5ΔNHA1 empty, pRS426 AVP1(YCplac111) empty, pRS423
115 ΔENA1,2,5ΔNHA1 NHA1 AVP1(YCplac111) 938 AtNHXS1(423)
116 ΔENA1,2,5ΔNHA1 NHA1 AVP1(YCplac111) 1301 SseNHX1(423)
117 ΔENA1,2,5ΔNHA1 NHA1 AVP1(YCplac111) 938 AtNHXS1(423)
118 ΔENA1,2,5ΔNHA1 NHA1 AVP1(YCplac111) 1301 SseNHX1(423)
119 ΔENA1,2,5ΔNHA1 NHA1 AVP1(YCplac111) empty, pRS423
120 ΔENA1,2,5ΔNHA1 NHA1 AVP1(YCplac111) empty, pRS423
121 ΔENA1,2,5ΔNHA1 NHA1 AVP1(YCplac111) empty, pRS423