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<!------------ 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
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30 |
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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, Tamukaicomponent | 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, Shimazoesample | 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, Shimazoesample | 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, Tamukaicomponent | 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)
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% EtOH
preserve in -20℃
7/12
OE-PCR
Nambusample | 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)
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)
Add 0.6μl 10x Buffer to all sample (1~14)
Culture in 10mL
8/7
YPD
TongComponent | (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
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
NambuNumber | 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): 0.5µL
Insert: 1.0µl
Ligation High: 1.5µl
Put in Heatblock
8/13
YPD culture
TongNaCl 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
TamukaiSample Name | Sample Volume (μl) | Competent Cells/(μl) |
---|---|---|
Mangrin | 3 | 15 |
ZrFPS1-2 | 3 | 15 |
McHKT2 | 3 | 15 |
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
NambuMaterial | 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
TongNumber | 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.
mix the following material
SmaCIP (Backbone) : 1ul
gBlock (Insert) : 1ul
Ligation High : 2ul
(Total : 4ul)
putin HeatBlock overnight at 18℃
8/19
PCR
Nambusample | 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
Nambunumber | 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
NambuSample 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
Shimazoeplasmid | 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
Tongplasmid | 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
Shimazoeplasmid | 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
Shimazoesample | 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
Nambusample | 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
Miniprep_Nanodrop
Nambusample:505,506,507,508,516,522,530
98℃* 10s
50℃* 5s
68℃* 20s
68℃ 1min
4℃ ∞
(*× 40 cycle)
8/28
PCR
ShimazoeComponent | 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
ShimazoeComponent | 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
ShimazoeComponent | 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
TongNumber | 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
Tong100ml 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
ShimazoeComponent | 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
ShimazoeComponent | 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
Shimazoeplasmid | 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
Shimazoesample | 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
Shimazoetemplate | 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
Shimazoenumber | 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
Shimazoetemplate | 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)
・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
Shimazoesample | 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
ShimazoeNumber | 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
Shimazoetemplate | 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
Shimazoetemplate | 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
ShimazoeMaterial | 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)
on ice 20min
98℃ 10s
98℃* 10s
50℃* 5s
68℃* 30s
68℃ 30s
(*× 30cycle)
9/2
PCR
ShimazoeMaterial | 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
ShimazoeMaterial | 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
ShimazoeMaterial | 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
Tongsample | 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
on ice 20min
42℃ 45sec
on ice 3min
SOC 550
37℃ 40min
9/4
PCR
TongMaterial | 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
ShimazoeMaterial | 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
ShimazoeMaterial | 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
ShimazoeMaterial | 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
Tongnumber | 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
TongMaterial | 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
ShimazoeMaterial | 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
Shimazoe1 | 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
Shimazoetemplate | 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
ShimazoeMaterial | 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
TamukaiMaterial | 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)
・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
ShimazoeMaterial | 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
Shimazoeinsert | 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
Colony PCR
ShimazoeMaterial | 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
ShimazoeMaterial | 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
ShimazoeMaterial | 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
Tongplasmid | 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
Tongvolume(ul) | 10×H buffer(ul) | EcoRⅠ(ul) | PstⅠ(ul) | DW(ul) | Total/(ul) | |
---|---|---|---|---|---|---|
DNA 404 | 5 | 4 | 2 | 2 | 27 | 40 |
9/8
Ligation
Tongsample | 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
Tongtemplate | 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)
・on ice 10min
・42℃ 45sec
・+SOC 300ul
・37℃ 40min
9/8
OE-PCR
Tongtemplate① | 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
TongMaterial | 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
Tongvolume(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 |
9/9
OE-PCR
Tongtemplate | 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
Tongtemplate | 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
Tongtemplate① | 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)
on ice 20min
9/10
Colony PCR
TongMaterial | 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
TamukaiMaterial | 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
ShimazoeMaterial | 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
NambuMaterial | 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
Shimazoematerial | volume(μl) |
---|---|
EcoT14Ⅰ | 25 |
10×H buffer | 50 |
λDNA | 71 |
DW | 354 |
Total | 500 |
9/11
Restriction Enzyme Digestion
Nambumaterial | 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
Nambumaterial | 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
ShimazoeMaterial | 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
Shimazoeinsert | 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 |
NHA1
98℃ 2min
98℃* 30s
48℃* 30s
68℃* 3m30s
68℃ 5min
4℃ ∞
(*× 30 cycle)
9/11
PCR
Tamukaimaterial | 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
Tamukaimaterial | 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
Shimazoeinsert | 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
Nambumaterial | 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 |
material | volume(μl) |
---|---|
AVP1(on pSB1C3) | 5 |
BSA | 2 |
10×H buffer | 2 |
Nco1 | 1 |
EcoR1 | 1 |
Pst1 | 1 |
DW | 8 |
Total | 20 |
4sample(3,6,7,11)
9/12
Ligation
ShimazoeMaterial | Volume(ul) |
---|---|
Ligation high | 4 |
pRS425 Not1 CIAP | 2 |
AVP1 Not1 ③ | 2 |
Total | 8 |
9/13
OE-PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 30 |
10uM Z017℗ | 1.2 |
10uM Z018℗ | 1.2 |
template⓪ | 6 |
template① | 6 |
template② | 6 |
Tks Gflex DNA Polymerase | 1.2 |
DW | 8.4 |
Total | 60 |
ZrGPD1
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 30 cycle)
9/13
OE-PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Prime Star Max | 5 |
Z047 | 1 |
Z074 or Z048 | 1 |
template | 1 |
DW | 2 |
Total | 10 |
98℃* 10s
55℃* 15s
68℃* 10s
(*× 35 cycle)
9/13
PCR
Shimazoematerial | volume (ul) |
---|---|
2× Prime Star Max | 5 |
10uM Z047 | 1 |
10uM Z074 | 1 |
template | 0.5 |
DW | 2.5 |
Total | 10 |
9/13
Colony PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 10 |
10uM m13f | 0.2 |
10uM m13r | 0.2 |
DW | 9.6 |
Total | 20 |
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 2min
72℃ 2min
4℃ ∞
(*× 30cycle)
9/13
OE-PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 5 |
10uM Z044 | 0.2 |
10uM Z047 | 0.2 |
template① | 0.5 |
template② | 0.5 |
Tks Gflex DNA Polymerase | 0.1 |
DW | 3.5 |
Total | 10 |
Stalk-SdrG, Stalk-FgβF3
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 2min
68℃ 2min
4℃ ∞
(*× 30 cycle)
9/14
Restriction Enzyme Digestion
Nambumaterial | volume(μl) |
---|---|
sample(404/910/938) | 10/5/3 |
EcoR1 | 2 |
10×H buffer | 4 |
Pst1 | 2 |
DW | 22/27/32 |
Total | 40 |
9/14
Colony PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 62.5 |
10uM m13f | 1.25 |
10uM m13r | 1.25 |
DW | 60 |
Total | 125 |
SseNHX1/AVP1
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 2min
72℃ 2min
4℃ ∞
(*× 30cycle)
9/14
Restriction Enzyme Digestion
Nambusample | 404 | 910 | 938 |
---|---|---|---|
volume(ul) | 10 | 5 | 3 |
10×H buffer(10xH) | 4 | 4 | 4 |
EcoR1 | 2 | 2 | 2 |
Not1 | 2 | 2 | 2 |
DW | 22 | 27 | 32 |
Total | 40 | 40 | 43 |
9/15
OE-PCR
TongMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 30 |
10uM Z017℗ | 1.2 |
10uM Z018℗ | 1.2 |
template pro | 6 |
template term | 6 |
template ORF | 6 |
Tks Gflex DNA Polymerase | 1.2 |
DW | 8.4 |
Total | 60 |
ZrGPD1
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 30 cycle)
98℃* 10s
55℃* 15s
72℃* 10s
(*× 35 cycle)
9/15
OE-PCR
Tongnumber | gene(plasmid) | strain | marker |
---|---|---|---|
1 | ×(pRS426) | WT | URA3 |
2 | AtHKT1(pRS426) | WT | URA3 |
3 | Mangrin(pRS316) | G19 | URA3 |
4 | ×(pRS316) | G19 | URA3 |
5 | ×(pRS316) | WT | URA3 |
6 | Mangrin(pRS316) | WT | URA3 |
9/15
OE-PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 25 |
10uM Z017℗ | 1 |
10uM Z043 | 1 |
template① | 3 |
template② | 3 |
Tks Gflex DNA Polymerase | 1 |
DW | 16 |
Total | 50 |
ZrGPD1
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
9/15
Restriction Enzyme Digestion
Nambumaterial | volume(μl) |
---|---|
ZrFPS1-1st | 7 |
EcoR1 | 0.5 |
Nco1 | 0.5 |
10×K buffer | 1 |
BSA | 1 |
Total | 10 |
material | volume(μl) |
---|---|
ZrFPS1-2nd (718) | 4 |
Spe1 | 2 |
Nco1 | 2 |
10×K buffer | 4 |
BSA | 4 |
DW | 24 |
Total | 40 |
material | volume(μl) |
---|---|
pRS426 | 2 |
EcoR1 | 2 |
Spe1 | 2 |
10×H buffer | 4 |
CIAP | 1 |
DW | 29 |
Total | 40 |
material | volume(μl) |
---|---|
pRS316 | 2 |
EcoR1 | 2 |
Spe1 | 2 |
10×H buffer | 4 |
CIAP | 1 |
DW | 29 |
Total | 40 |
Stalk
98℃* 10s
55℃* 15s
72℃* 10s
4℃ ∞
(*× 30 cycle)
9/16
PCR
TongMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 30 |
10uM Z017℗ | 1.2 |
10uM Z018℗ | 1.2 |
template⓪ | 6 |
template① | 6 |
template② | 6 |
Tks Gflex DNA Polymerase | 1.2 |
DW | 8.4 |
Total | 60 |
ZrGPD1
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 35 cycle)
9/16
PCR
Tongmaterial | volume (ul) |
---|---|
2× Prime Star Max | 5 |
10uM Z047 | 1 |
10uM Z074 | 1 |
template | 0.5 |
DW | 2.5 |
Total | 10 |
Stalk
9/16
PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 25 |
10uM primer① | 1 |
10uM primer② | 1 |
template | 1 |
Tks Gflex DNA Polymerase | 1 |
DW | 21 |
Total | 50 |
ZrGPD1
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 30 cycle)
9/16
OE-PCR
Tongmaterial | volume (ul) |
---|---|
2× Prime Star Max | 20 |
10uM Z017℗ | 1 |
10uM Z018℗ | 1 |
promoter | 3 |
SseNHX1 | 3 |
terminator | 3 |
DW | 9 |
Total | 40 |
9/16
Restriction Enzyme Digestion
Nambusample | 1203(RFP) | 1204(GFP) |
---|---|---|
volume(ul) | 2 | 1 |
10×H buffer(10xH) | 2 | 2 |
EcoR1 | 1 | 1 |
Not1 | 1 | 1 |
DW | 14 | 15 |
Total | 20 | 20 |
9/17
OE-PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 30 |
10uM Z017℗ | 1.2 |
10uM Z018℗ | 1.2 |
template⓪ | 6 |
template① | 6 |
template② | 6 |
Tks Gflex DNA Polymerase | 1.2 |
DW | 8.4 |
Total | 60 |
ZrGPD1
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 30 cycle)
9/17
PCR
Shimazoematerial | volume (ul) |
---|---|
5× Q5 Reaction Buffer | 5 |
10uM dNTPs | 0.5 |
Q5 DNA Polymerase | 0.25 |
DW | 15.75 |
10uM primer① | 1.25 |
10uM primer② | 1.25 |
template | 1 |
Total | 25 |
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
ShimazoeMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 12.5 |
10uM primer① | 1 |
10uM primer② | 1 |
template | 1 |
Tks Gflex DNA Polymerase | 1 |
DW | 8.5 |
Total | 25 |
pro,term,Sse ORF
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 30 cycle)
9/17
Transformation
Nambuvolume(ul) | Competent Cells(ul) | Total(ul) | |
---|---|---|---|
DNA J04450 | 1 | 10 | 11 |
volume(ul) | Competent Cells(ul) | Total(ul) | |
---|---|---|---|
DNA | 0.5 | 10 | 10.5 |
DNA J04450…2017 Kit Plate 1 24A
9/18
OE-PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 30 |
10uM Z017℗ | 1.2 |
10uM Z018℗ | 1.2 |
template⓪ | 3 |
template① | 3 |
template② | 3 |
Tks Gflex DNA Polymerase | 1.2 |
DW | 17.4 |
Total | 60 |
ZrGPD1,SseNHX1
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 30 cycle)
9/18
Ligation
TongMaterial | Volume(ul) |
---|---|
Ligation high | 2 |
pRS425 | 1 |
AVP1(3) | 1 |
Total | 4 |
Material | Volume(ul) |
---|---|
Ligation high | 3.5 |
pRS425 | 0.5 |
AVP1(3) | 3 |
Total | 7 |
9/18
Inverse PCR
Shimazoematerial | volume (ul) |
---|---|
2× Prime Star Max | 25 |
10uM Z039 | 1 |
10uM Z040 | 1 |
template(938) | 0.5 |
DW | 22.5 |
Total | 50 |
98℃* 10s
55℃* 5s
72℃* 40s
(*× 30 cycle)
9/18
SLiCE
ShimazoeMaterial | Volume(ul) |
---|---|
vector | 0.5 |
insert | 1 |
10× SLiCE Buffer | 1 |
SLiCE | 1 |
DW | 6.5 |
Total | 10 |
SseNHX1/GPD1
Material | Volume(ul) |
---|---|
vector | 0.5 |
insert① | 1 |
insert② | 1 |
10× SLiCE Buffer | 1 |
SLiCE | 1 |
DW | 5.5 |
Total | 10 |
Fgβ/SdrG
9/18
Transformation
ShimazoeLigation products | volume(ul) | Competent Cells/(ul) | Total(ul) |
---|---|---|---|
SseNHX1/GPD1 | 10 | 50 | 60 |
Fgβ/SdrG | 10 | 50 | 60 |
9/19
OE-PCR
TongMaterial | Volume(ul) |
---|---|
2× Gflex buffer | 12.5 |
Z044 | 0.5 |
Z047 | 0.5 |
SdrG or Fgβ | 1 |
Stalk | 1 |
Gflex DNA polymerase | 0.5 |
DW | 9 |
Total | 25 |
9/19
Colony PCR
TongMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 82.5 |
10uM m13f | 1.65 |
10uM m13r | 1.65 |
DW | 79.2 |
Total | 165 |
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 4min
72℃ 4min
4℃ ∞
(*× 30cycle)
9/19
Inverse PCR
Tongmaterial | volume (ul) |
---|---|
2× Prime Star Max | 25 |
10uM Z039 | 1 |
10uM Z040 | 1 |
template(938) | 0.6 |
DW | 22.5 |
Total | 50 |
98℃* 10s
55℃* 5s
72℃* 40s
(*× 30cycle)
9/19
PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 25 |
10uM primer① | 1 |
10uM primer② | 1 |
template | 1 |
Tks Gflex DNA Polymerase | 1 |
DW | 21 |
Total | 50 |
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
TongMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 17.5 |
10uM Z047 | 0.35 |
10uM Z074 | 0.35 |
DW | 16.8 |
Total | 35 |
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 3min
72℃ 2min
4℃ ∞
(*× 30cycle)
37℃/1h
9/20
Inverse PCR
Tongmaterial | volume (ul) |
---|---|
2× Prime Star Max | 25 |
10uM Z039 | 1 |
10uM Z040 | 1 |
template(938) 1/200 | 0.6 |
DW | 22.4 |
Total | 50 |
98℃* 10s
55℃* 5s
72℃* 40s
(*× 30cycle)
9/20
Restriction Enzyme Digestion
TongDpn1 digestion
9/20
PCR
Tongmaterial | volume (ul) |
---|---|
2× Prime Star Max | 25 |
10uM Z047 | 1 |
10uM Z074 | 1 |
template①/② | 1/1 |
DW | 22 |
Total | 50 |
98℃* 10s
55℃* 15s
72℃* 10s
4℃ ∞
(*× 30cycle)
9/20
Ligation
ShimazoeMaterial | Volume(ul) |
---|---|
Ligation high | 2 |
vector | 1 |
insert | 1 |
Total | 4 |
Mangrin : pRS426
AVP1 : pRS425
9/20
Restriction Enzyme Digestion
Nambusample | 1 | 2 | 3 | 4 |
---|---|---|---|---|
volume(ul) | 6 | 6 | 6 | 9 |
10×H buffer(10xH) | 3 | 3 | 3 | 3 |
EcoR1 | 1.5 | 1.5 | 1.5 | 1.5 |
Pst1 | 1.5 | 1.5 | 1.5 | 1.5 |
DW | 18 | 18 | 18 | 15 |
Total | 30 | 30 | 30 | 30 |
put in incubator at 37℃
9/21
Qubit^tm dsDNA HS Assay Kit
ShimazoeMaterial | Volume(ul) |
---|---|
dsDNA buffer | 200 |
dsDNA reagent | 1 |
dsDNA solution | 1 |
Total | 202 |
9/21
Ligation
ShimazoeMaterial | Volume(ul) |
---|---|
Ligation high | 6 |
vector 426/316 | 2/2 |
ZrFPS1-1st | 2 |
ZrFPS1-2nd | 2 |
Total | 12 |
9/21
SLiCE
ShimazoeMaterial | Volume(ul) |
---|---|
DW | 5.5 |
pIEG-SSS 1/20 | 1 |
FgβF3 1/10 | 1 |
Stalk | 0.5 |
10× SLiCE Buffer | 1 |
SLiCE | 1 |
Total | 10 |
Material | Volume(ul) |
---|---|
DW | 6.1 |
pIEG-SSS 1/20 | 1 |
SdrG | 0.4 |
Stalk | 0.5 |
10× SLiCE Buffer | 1 |
SLiCE | 1 |
Total | 10 |
9/21
PCR
Shimazoematerial | volume (ul) |
---|---|
2× Prime Star Max | 12.5 |
10uM Z041 | 1 |
10uM Z042 | 1 |
template 1/200 | 1 |
DW | 9.5 |
Total | 25 |
template : 317(SseNHX1), 506(ZrGPD1)
98℃* 10s
55℃* 5s
72℃* 10s
(*× 30cycle)
9/21
PCR
Shimazoematerial | volume (ul) |
---|---|
2× Prime Star Max | 25 |
10uM Z041 | 2 |
10uM Z042 | 2 |
template 1/200 | 1 |
DW | 20 |
Total | 50 |
template : 317(SseNHX1), 506(ZrGPD1)
98℃* 10s
55℃* 5s
72℃* 10s
(*× 30cycle)
template(SseNHX1/ZrGPD1/Stalk)
on ice 30min
42℃45s
on ice 2min
SOC 500ul
37℃ 40min
9/21
Transformation
Tongyeast | plasmid | |
---|---|---|
⑦ | WT(547) | ・AVP1(YCplac111) ・AtNHXS1(pRS423)(938) ・empty(pRS316) |
⑧ | WT | ・AVP1(YCplac111) ・AtNHXS1(pRS313)(938) ・empty(pRS316) |
⑨ | WT | ・AVP1(YCplac111) ・empty(pRS313) ・empty(pRS316) |
⑩ | WT | ・AtNHXS1(pRS423)(938) ・Mangrin(pRS316) |
⑪ | WT | ・empty(YCplac111) ・AtNHXS1(pRS313) ・empty(pRS316) |
⑫ | ΔENA1(559) | ・AVP1(YCplac111) ・AtNHXS1(pRS423) ・empty(pRS316) |
competent cell : 10ul
plasmid : 0.5ul
Solution3 : 100ul
vortex 30s
37℃/35min
9/22
SLiCE
ShimazoeMaterial | Volume(ul) |
---|---|
DW | 5.5 |
pIEG-SSS 1/20 | 1 |
FgβF3 1/10 | 1 |
Stalk | 0.5 |
10× SLiCE Buffer | 1 |
SLiCE | 1 |
Total | 10 |
Material | Volume(ul) |
---|---|
DW | 5 |
pIEG-SSS 1/20 | 1 |
SdrG 1/4 | 1 |
Stalk 1/2 | 1 |
10× SLiCE Buffer | 1 |
SLiCE | 1 |
Total | 10 |
9/22
In Fusion
ShimazoeMaterial | Volume(ul) |
---|---|
DW | 5 |
pIEG-SSS 1/20 | 1 |
FgβF3 1/10 | 1 |
Stalk | 0.5 |
5× In-Fusion HD Enzyme Premix | 2 |
Total | 10 |
Material | Volume(ul) |
---|---|
DW | 5 |
pIEG-SSS 1/20 | 1 |
SdrG 1/4 | 1 |
Stalk 1/2 | 1 |
5× In-Fusion HD Enzyme Premix | 2 |
Total | 10 |
9/22
Colony PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 25 |
10uM m13f | 0.5 |
10uM m13r | 0.5 |
DW | 24 |
Total | 50 |
Material | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 15 |
10uM Z044 | 0.3 |
10uM Z047 | 0.3 |
DW | 14.4 |
Total | 30 |
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 3min
72℃ 2min
4℃ ∞
(*× 30cycle)
9/22
PCR
TongMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 8 |
10uM dNTPs | 3.2 |
10uM Z005 | 1 |
10uM Z028 | 1 |
template | 1 |
enzyme | 0.4 |
DW | 25.4 |
Total | 40 |
Cassette for ΔENA1,2,5
98℃* 10s
55℃* 15s
72℃* 2min
(*× 30 cycle)
9/22
Spot
TongNumber | Material< /th> |
---|---|
① | 547 WT |
② | 554 ΔNHA1 |
③ | 555 G19 |
④ | 557 ΔNHA1,ΔENA1 |
⑤ | 559 ΔENA1 |
⑥ | ΔENA1.2.5, ΔNHA1 |
⑦ | 564 Z.roxii |
Cassette for ΔENA1,2,5
98℃* 10s
55℃* 15s
72℃* 2min
(*× 30 cycle)
9/22
Restriction Enzyme Digestion
Nambusample | 1087(AtNHXS1) | 910(AtHKT1) |
---|---|---|
volume(ul) | 20 | 20 |
10×H buffer(10xH) | 4 | 4 |
EcoR1 | 1 | 1 |
Not1 | 1 | 1 |
DW | 14 | 14 |
Total | 40 | 40 |
enzyme inactivation 60℃ 15min
9/22
Restriction Enzyme Digestion
Nambusample | pSB1C3 |
---|---|
volume(ul) | 20 |
10×H buffer(10xH) | 4 |
EcoR1 | 2 |
Not1 | 2 |
Dpu1 | 2 |
DW | 10 |
Total | 40 |
enzyme inactivation 80℃ 20min
9/23
Colony PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 35 |
10uM m13f | 0.7 |
10uM m13r | 0.7 |
DW | 33.6 |
Total | 70 |
Material | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 85 |
10uM Z044 | 1.7 |
10uM Z047 | 1.7 |
DW | 81.6 |
Total | 170 |
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 3min
72℃ 2min
4℃ ∞
(*× 30cycle)
9/23
PCR
TongMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 8 |
10uM dNTPs | 3.2 |
10uM Z003 | 1 |
10uM Z004 | 1 |
Yeast Genome | 1 |
enzyme | 0.4 |
DW | 25.4 |
Total | 40 |
Cassette for ΔENA1,2,5
98℃* 10s
55℃* 15s
72℃* 4min
(*× 35 cycle)
9/23
Restriction Enzyme Digestion
Tongmaterial | volume (ul) |
---|---|
Plasmid(404,716,910,3) | 10 |
10×H buffer | 2 |
BSA | 2 |
Triton X-100 | 2 |
Not1 | 1 |
DW | 3 |
Total | 20 |
404,716: Mangrin(pRS716)
910: AtHKT1(pRS426)
3: AVP1(pSB1C3)
9/23
Colony PCR
NambuMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 18.75 |
10uM m13f | 0.375 |
10uM m13r | 0.375 |
DW | 18 |
Total | 37.5 |
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 3min
72℃ 2min
4℃ ∞
(*× 30cycle)
9/24
Colony PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 120 |
10uM Z044 | 2.4 |
10uM Z047 | 2.4 |
DW | 115.2 |
Total | 240 |
9/24
Ligation
Nambunumber | insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
---|---|---|---|---|---|---|
1.2.1 | 910(heat) | 1.5 | 1.2 | 0.5 | 2 | 4 |
1.2.2 | 910(column) | 1.5 | 1.2 | 0.5 | 2 | 4 |
1.2.3 | 1087(heat) | 1.5 | 1.2 | 0.5 | 2 | 4 |
1.2.4 | 1087(column) | 1.5 | 1.2 | 0.5 | 2 | 4 |
PL1 | 910(heat) | 1.5 | pSB1C3 | 0.5 | 2 | 4 |
PL2 | 910(column) | 1.5 | pSB1C3 | 0.5 | 2 | 4 |
PL3 | 1087(heat) | 1.5 | pSB1C3 | 0.5 | 2 | 4 |
PL4 | 1087(column) | 1.5 | pSB1C3 | 0.5 | 2 | 4 |
DNA(1.2.1/1.2.2/1.2.3/1.2.4/PL1/PL2/PL3/PL4)
9/25
Ligation
ShimazoeMaterial | Volume(ul) |
---|---|
Ligation high | 2 |
insert | 1 |
vector | 1 |
Total | 4 |
① | ② | ③ | ④ | ⑤ | ⑥ | ⑦ | ⑧ | ⑨ | |
---|---|---|---|---|---|---|---|---|---|
insert | 404 | 716 | 1304 | 1306 | 1310 | 1118 | 1301 | 1302 | 1303 |
vector | 426 | 426 | 313 | 313 | 313 | 425 | 313 | 313 | 313 |
9/25
Colony PCR
NambuMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 75 |
10uM m13f | 1.5 |
10uM m13r | 1.5 |
DW | 72 |
Total | 150 |
AtNHXS1(pSB1C3)/AtHKT1(pRS426)/McHKT2(pRS426,pRS316)
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 3min
72℃ 2min
4℃ ∞
(*× 30cycle)
9/25
Restriction Enzyme Digestion
Nambusample | DNA(pSB1C3 Linear) |
---|---|
volume(ul) | 5 |
10×M buffer | 2 |
Xba1 | 1 |
Pst1 | 1 |
Dpn1 | 1 |
DW | 10 |
Total | 20 |
enzyme inactivation 80℃ 20min
9/25
Restriction Enzyme Digestion
Nambusample | 1301 | 1302 | 1303 | 1304 | 1306 | 1310 |
---|---|---|---|---|---|---|
volume(ul) | 10 | 10 | 10 | 10 | 10 | 2 |
10×H buffer | 2 | 2 | 2 | 2 | 2 | 2 |
EcoR1 | 1 | 1 | 1 | 1 | 1 | 1 |
Pst1 | 1 | 1 | 1 | 1 | 1 | 1 |
DW | 6 | 6 | 6 | 6 | 6 | 14 |
Total | 20 | 20 | 20 | 20 | 20 | 20 |
enzyme inactivation 60℃ 15min
9/26
Colony PCR
ShimazoeMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 36 |
10uM dNTPs | 3.6 |
10uM m13r | 9 |
10uM m13f | 9 |
Q5 DNA Polymerase | 1.8 |
DW | 120.5 |
Total | 180 |
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30cycle)
9/26
PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 12.5 |
10uM primer① | 0.5 |
10uM primer② | 0.5 |
template | 0.5 |
Tks Gflex DNA Polymerase | 0.5 |
DW | 10.5 |
Total | 25 |
ZrFPS1-1st | ZrFPS1-2nd | |
---|---|---|
primer | Z041, Z043 | Z077, Z042 |
template | 927 | 718 |
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1min
68℃ 2min
4℃ ∞
(*× 30 cycle)
9/26
Restriction Enzyme Digestion
Nambusample | Plasmid(AVP1.pSB1C3) |
---|---|
volume(ul) | 20 |
10×H buffer | 4 |
EcoR1 | 1 |
Pst1 | 1 |
CIAP | 1 |
DW | 13 |
Total | 40 |
9/27
Transformation(yeast)
Tongyeast | plasmid | |
---|---|---|
34 | WT | ・empty(pRS425) ・empty(pRS316) ・Mangrin(pRS316) |
35 | WT | ・AVP1(YCplac111) ・SseNHX1(pRS423) ・empty(pRS316) |
36 | WT | ・AVP1(YCplac111) ・AtNHXS1 (pRS423) ・Mangrin(pRS316) |
37 | WT | ・AVP1(YCplac111) ・AtNHXS1(pRS313) ・Mangrin(pRS316) |
38 | WT | ・AVP1(YCplac111) ・(1301) SseNHX1(pRS423) ・Mangrin(pRS316) |
39 | WT | ・empty(pRS425) ・(1432)SdrG(pIEG-SSS:HIS) ・(1204)GFP(pRS316) |
40 | WT | ・empty(pRS425) ・(1444)SdrG ・(1204)GFP |
41 | WT | ・empty(pRS425) ・(1448)SdrG ・(1204)GFP(pRS316) |
9/28
OD measure
Tongnumber | OD | number | OD | |
---|---|---|---|---|
7 | 4.8 | 20 | 6.6 | |
8 | 5.8 | 21 | 6.2 | |
9 | 6.4 | 22 | 5.9 | |
10 | 6.8 | 23 | × | |
11 | 6.6 | 24 | × | |
12 | 6.0 | 25 | × | |
13 | 6.6 | 26 | × | |
14 | 6.4 | 27 | × | |
15 | × | 28 | 4.1 | |
16 | 6.6 | 29 | 4.7 | |
17 | 6.9 | 30 | 3.8 | |
18 | 6.6 | 31 | /RFP | |
19 | 6.4 | 32 | /GFP |
9/28
PCR
Shimazoematerial | volume (ul) |
---|---|
2× Prime Star Max | 12.5 |
10uM primer① | 1 |
10uM primer② | 1 |
template | 0.5 |
DW | 10 |
Total | 25 |
Fgβ | SdrG | stalk for Fgβ | stalk for SdrG | |
---|---|---|---|---|
template | 342, 1/300 | 752, 1/100 | gBlock | gBlock |
primer | Z086 Z084 | Z086 Z045 | Z046 Z087 | Z074 Z087 |
9/28
Inverse PCR
Shimazoematerial | volume (ul) |
---|---|
2× Prime Star Max | 25 |
10uM Z085 | 2 |
10uM Z088 | 2 |
template(pIEG-SSS) | 1 |
DW | 20 |
Total | 50 |
98℃* 10s
55℃* 5s
72℃* 40s
(*× 30 cycle)
9/28
OE-PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 25 |
10uM Z041 | 1 |
10uM Z042 | 1 |
ZrFPS1-1st | 1 |
ZrFPS1-2nd | 1 |
Tks Gflex DNA Polymerase | 1 |
DW | 20 |
Total | 50 |
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 2min
68℃ 2min
4℃ ∞
(*× 30 cycle)
9/28
PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 25 |
10uM primer① | 1 |
10uM primer② | 1 |
template | 1 |
Tks Gflex DNA Polymerase | 1 |
DW | 21 |
Total | 50 |
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 2min
68℃ 2min
4℃ ∞
(*× 30 cycle)
9/28
Restriction Enzyme Digestion
Nambusample | Mangrin | AVP1・pSB1C3 |
---|---|---|
volume(ul) | 20 | 20 |
10×H buffer | 4 | 4 |
EcoR1 | 1 | 1 |
Pst1 | 1 | 1 |
CIAP | × | 1 |
DW | 14 | 13 |
Total | 40 | 40 |
9/28
PCR
NambuStalk | FgβF3 | SdrG | AtHKT1(910) | |
---|---|---|---|---|
2× Prime Star Max (ul) | 20 | 20 | 20 | 20 |
F.primer | 1(Z082) | 1(Z080) | 1(Z078) | 1(Z094) |
R.primer | 1(Z083) | 1(Z081) | 1(Z079) | 1(Z095) |
template | 1(gBlock) | × | 0.5(752,1/100) | 1(910,1/100) |
DW | 17 | 18 | 17.5 | 17 |
Total | 40 | 40 | 40 | 40 |
9/28
Restriction Enzyme Digestion
Nambusample | 910-1 | 910-2 |
---|---|---|
volume(ul) | 20 | 20 |
10×M buffer | 4 | 4 |
Xba1 | 1 | 1 |
Pst1 | 1 | 1 |
DW | 14 | 14 |
Total | 40 | 40 |
9/29
Restriction Enzyme Digestion
Tongmaterial | volume (ul) |
---|---|
plasmid(716/1301/1304/938) | 10/15/15/15 |
10×H buffer | 4 |
EcoR1 | 0.5 |
Pst1 | 0.5 |
DW | 25/20/20/20 |
Total | 40 |
insert
material | volume (ul) |
---|---|
plasmid(6) | 20 |
SAP | 1 |
10×H buffer | 8 |
EcoR1 | 1 |
Pst1 | 1 |
DW | 49 |
Total | 80 |
Backbone
material | volume (ul) |
---|---|
plasmid(910) | 15 |
10×M buffer | 4 |
Xba1 | 0.5 |
Pst1 | 0.5 |
DW | 20 |
Total | 40 |
insert
material | volume (ul) |
---|---|
plasmid(6) | 10 |
SAP | 0.5 |
10×M buffer | 4 |
Xba1 | 0.5 |
Pst1 | 0.5 |
DW | 24.5 |
Total | 40 |
Backbone
9/29
PCR
TongMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 8 |
10uM dNTPs | 3.2 |
10uM primer① | 1 |
10uM primer② | 1 |
template | 1 |
enzyme | 0.4 |
DW | 25.4 |
Total | 40 |
SdrG | Fgβ | Stalk for Fgβ | Stalk for SdrG | |
---|---|---|---|---|
template | 752, 1/100 | 342, 1/300 | gBlock | gBlock |
primer | Z045, Z086 | Z084, Z086 | Z046, Z087 | Z074, Z087 |
9/29
OD measure
Tongnumber | OD | number | OD | number | OD | ||
---|---|---|---|---|---|---|---|
7-A | 1.13 | 11-A | 1.59 | 16-A | 0.54 | ||
7-B | 1.12 | 11-B | 1.57 | 16-B | 0.58 | ||
8-A | 1.24 | 12-A | 0.81 | 17-A | 1.49 | ||
8-B | 1.19 | 12-B | 0.80 | 17-B | 1.55 | ||
9-A | 1.11 | 13-A | 1.48 | 20-A | 0.87 | ||
9-B | 1.16 | 13-B | 1.44 | 20-B | 0.72 | ||
10-A | 1.59 | 14-A | 1.53 | 28-A | 1.13 | ||
10-B | 1.61 | 14-B | 1.52 | 28-B | 1.15 |
9/29
PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Gflex PCR Buffer | 20 |
10uM primer① | 1 |
10uM primer② | 1 |
template | 1 |
Tks Gflex DNA Polymerase | 1 |
DW | 16 |
Total | 40 |
Fgβ | SdrG | |
---|---|---|
template | 342 | 752 |
primer | Z089, Z090 | Z091, Z092 |
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 2min
68℃ 2min
4℃ ∞
(*× 30 cycle)
9/29
PCR
Shimazoematerial | volume (ul) |
---|---|
2× Prime Star Max | 12.5 |
10uM primer① | 1 |
10uM primer② | 1 |
template | 0.5 |
DW | 10 |
Total | 25 |
Stalk for Fgβ | Stalk for SdrG | Fgβ for OE-PCR | SdrG for OE-PCR | |
---|---|---|---|---|
template | gBlock | gBlock | 342 | gBlock |
primer | Z086 Z084 | Z074 Z087 | Z086 Z045 | Z093 Z087 |
98℃* 10s
55℃* 15s
72℃* 10s
(*× 30 cycle)
9/29
Restriction Enzyme Digestion
Nambusample | SdrG | Fgβ |
---|---|---|
volume(ul) | 34 | 34 |
10×H buffer | 4 | 4 |
EcoR1 | 1 | 1 |
Pst1 | 1 | 1 |
Total | 40 | 40 |
37℃ 3h
9/29
PCR
NambuMaterial | Volume(ul) |
---|---|
2× Prime Star Max (ul) | 12.5 |
F.primer (Z082) | 1 |
R.primer (Z083) | 1 |
template (gBlock) | 1 |
DW | 9.5 |
Total | 25 |
98℃* 10s
55℃* 5s
72℃* 11s
(*× 30cycle)
9/29
Ligation
Nambuinsert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
---|---|---|---|---|---|
Mangrin | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
ZrGPD1(1304) | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
SseNHX1(1302) | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
Fgβ | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
SdrG | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
AtNHXS1(1087) | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
Control(MilliQ) | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
9/29
Transformation
Nambuvolume(ul) | Competent Cells(ul) | Total(ul) | |
---|---|---|---|
Mangrin | 2 | 10 | 12 |
ZrGPD1(1304) | 2 | 10 | 12 |
SseNHX1(1302) | 2 | 10 | 12 |
Fgβ | 2 | 10 | 12 |
SdrG | 2 | 10 | 12 |
AtNHXS1(1087) | 2 | 10 | 12 |
Control(MilliQ) | 2 | 10 | 12 |
20℃ 40h
9/29
PCR
TongMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 8 |
10uM dNTPs | 3.2 |
10uM primer① | 1 |
10uM primer② | 1 |
template | 1 |
enzyme | 0.4 |
DW | 25.4 |
Total | 40 |
Fgβ | Stalk for Fgβ | Stalk for Fgβ | ||
---|---|---|---|---|
template | 342, 1/300 | gBlock,1/10 | gBlock,1/10 | |
primer | Z045, Z086 | Z046, Z087 | Z093, Z087 |
SdrG | Fgβ | Stalk for SdrG | |
---|---|---|---|
template | 752, 1/100 | 342, 1/300 | gBlock |
primer | Z045, Z086 | Z084, Z086 | Z074, Z087 |
9/29
Ligation
Tonginsert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
---|---|---|---|---|---|
AVP1・pSB1C3(E,P cut) | 1 | 1301 | 1 | 2 | 4 |
AVP1・pSB1C3(E,P cut) | 1 | 1304 | 1 | 2 | 4 |
AVP1・pSB1C3(E,P cut) | 1 | 716 | 1 | 2 | 4 |
AVP1・pSB1C3(E,P cut) | 1 | 938 | 1 | 2 | 4 |
insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
---|---|---|---|---|---|
AVP1・pSB1C3(E,P cut) | 1 | 910 | 3 | 4 | 8 |
9/30
PCR
TongMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 8 |
10uM dNTPs | 3.2 |
10uM primer① | 1 |
10uM primer② | 1 |
template | 1 |
enzyme | 0.4 |
DW | 25.4 |
Total | 40 |
number | ① | ② | ③ | ④ | ⑤ | ⑥ | ⑦ |
---|---|---|---|---|---|---|---|
template | 342, 1/300 | Stalk gBlock 1/10 | Stalk gBlock 1/10 | 13 gBlock | 13 gBlock | 13 1/50 | 13 1/50 |
primer | Z045, Z086 | Z046, Z087 | Z093, Z087 | Z046, Z087 | Z093, Z087 | Z046, Z087 | Z093, Z087 |
9/30
OE-PCR
TongMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 8 |
10uM dNTPs | 3.2 |
10uM primerF | 1 |
10uM primerR | 1 |
template 1 | 1 |
template 2 | 1 |
enzyme | 0.4 |
DW | 24.6 |
Total | 40 |
number | Template1 | Template2 | Primer |
---|---|---|---|
1 | Z045, Z086 | Z074, Z087 | Z086, Z087 |
2 | Z084, Z086 | Z074, Z087 | Z086, Z087 |
98℃* 10s
55℃* 5s
72℃* 2m30s
(*× 30cycle)
9/30
OE-PCR
TongMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 8 |
10uM dNTPs | 3.2 |
10uM primer① | 1 |
10uM primer② | 1 |
template 1 | 1 |
template 2 | 1 |
enzyme | 0.4 |
DW | 24.4 |
Total | 40 |
template2: ①Z046-Z087, ②Z093-Z087
on ice 15min/42℃ 45s/on ice 1min
10/1
Colony PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 90 |
10uM m13f | 1.8 |
10uM m13r | 1.8 |
DW | 86.4 |
Total | 180 |
10/1
Restriction Enzyme Digestion
Nambusample | Stalk | AVP1・pSB1C3 |
---|---|---|
volume(ul) | 19 | 10 |
10×H buffer | 3 | × |
10×M buffer | × | 2 |
Xba1 | × | 1 |
EcoR1 | 1 | × |
Pst1 | 1 | 1 |
DW | 6 | 7 |
Total | 30 | 21 |
10/1
Colony PCR
NambuMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 162.5 |
10uM m13f | 3.25 |
10uM m13r | 3.25 |
DW | 156 |
Total | 325 |
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 3min
72℃ 2min
15℃ ∞
(*× 30cycle)
10/1
Colony PCR
TongMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 2.5 |
VF2 | 0.1 |
VR | 0.1 |
DW | 2.3 |
Total | 5 |
Material | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 2.5 |
VF2 | 0.1 |
Z027 | 0.1 |
DW | 2.3 |
Total | 5 |
10/1
PCR
TongMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 8 |
10uM dNTPs | 3.2 |
Z086 | 1 |
Z087 | 1 |
template 1 | 1 |
template 2 | 1 |
enzyme | 0.4 |
DW | 24.4 |
Total | 40 |
number | 1 | 2 | 3 | 4 |
---|---|---|---|---|
template 1 | Z045, Z086 | Z045, Z086 | Z084, Z086 | Z084, Z086 |
template 2 | Z046, Z087 | Z093, Z087 | Z046, Z087 | Z093, Z087 |
10/1
In Fusion
ShimazoeMaterial | Volume(ul) |
---|---|
DW | 5 |
pIEG-SSS 1/3 | 1 |
Fgβ 1/10 | 1 |
Stalk for Fgβ1/2 | 1 |
5× In-Fusion HD Enzyme Premix | 2 |
Total | 10 |
Material | Volume(ul) |
---|---|
DW | 5 |
pIEG-SSS 1/3 | 1 |
SdrG 1/4 | 1 |
Stalk for SdrG | 1 |
5× In-Fusion HD Enzyme Premix | 2 |
Total | 10 |
10/1
Colony PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 160 |
10uM Z086 | 3.2 |
10uM Z087 | 3.2 |
DW | 153.6 |
Total | 320 |
98℃ 10s
98℃* 10s
60℃* 30s
72℃* 3min
72℃ 3min
(*× 30cycle)
10/2
Colony PCR
NambuMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 18.75 |
10uM m13f | 0.375 |
10uM m13r | 0.375 |
DW | 18 |
Total | 37.5 |
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m45s
72℃ 2min
15℃ ∞
(*× 30cycle)
10/2
Colony PCR
NambuMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 150 |
10uM m13f | 3 |
10uM m13r | 3 |
DW | 144 |
Total | 300 |
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
NambuMangrin(4) | ZrGPD1(12) | AtNHXS1(32) | Fgβ(33) | SdrG(44) | AtHKT1(910) |
---|---|---|---|---|---|
267.7 ng/ul | 211.6 ng/ul | 243.5 ng/ul | 222.1 ng/ul | 250.8 ng/ul | 23.7 ng/ul |
10/2
Colony PCR
Tong1~14 | 716(Mangrin) | pRS316 | 1516 | 3,8,14 |
15~26 | 1304(ZrGPD1) | pRS423 | 2501 | 19,20,22,24,26 |
27~42 | 938(AtNHXS1) | pRS423 | 2024 | 30,31,33 |
43~54,66,71 | 1301(SseNHX1) | pRS423 | 2957(VR,VF2)/2811(VF2,Z027) | 40,41,44,47 |
55~65,67,(68),69,70 | 910(AtHKT1) | pRS423 | 2670(VF2,Z027)/2816(VR,VF) | 55,63,67,68,69,70 |
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
TongMaterial | Volume | |
---|---|---|
5× Q5 Reaction Buffer | 8 | |
10uM dNTPs | 3.2 | |
10uM primerF | 1 | |
10uM primerR | 1 | |
template | 1 | |
enzyme | 0.4 | |
DW | 25.4 | |
Total | 40 |
① | ② | |
---|---|---|
template | 752, 1/100 | gBlock, 1/10 |
primer | Z078, Z079 | Z082, Z083 |
SdrG, Stalk
10/2
PCR
TongMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 8 |
10uM dNTPs | 3.2 |
10uM primerF(Z080) | 1 |
10uM primerR(Z081) | 1 |
enzyme | 0.4 |
DW | 26.4 |
Total | 40 |
Fgβ
10/2
Restriction Enzyme Digestion
Tongsample | 3/8/14/19/20/22/24/26/30/31/33/40/41/44/47 |
---|---|
volume(ul) | 10 |
10×H buffer | 4 |
EcoR1 | 0.5 |
Pst1 | 0.5 |
DW | 25 |
Total | 40 |
10/2
PCR
ShimazoeMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 4 |
10uM dNTPs | 1.6 |
10uM Z094 | 0.5 |
10uM Z039 | 0.5 |
template(p079) | 0.5 |
DW | 12.7 |
PrimeSTAR HS | 0.2 |
Total | 20 |
Material | Volume |
---|---|
5× Q5 Reaction Buffer | 4 |
10uM dNTPs | 1.6 |
10uM Z095 | 0.5 |
10uM Z077 | 0.5 |
template(718) | 0.5 |
DW | 12.7 |
PrimeSTAR HS | 0.2 |
Total | 20 |
Fgβ
10/3
Restriction Enzyme Digestion
Nambusample | Mangrin(4) | ZrGPD1(12) | AtNHXS1(32) | Fgβ(33) | SdrG(44) |
---|---|---|---|---|---|
volume(ul) | 5 | 5 | 5 | 5 | 5 |
10×H buffer | 2 | 2 | 2 | 2 | 2 |
EcoR1 | 1 | 1 | 1 | 1 | 1 |
Pst1 | 1 | 1 | 1 | 1 | 1 |
DW | 11 | 11 | 11 | 11 | 11 |
Total | 20 | 20 | 20 | 20 | 20 |
sample | AtHKT1 | AVP1・pSB1C3 |
---|---|---|
volume(ul) | 30 | 20 |
10×M buffer | 4 | 3 |
Xba1 | 1 | 1 |
Pst1 | 1 | 1 |
CIAP | × | 1 |
DW | 4 | 4 |
Total | 40 | 30 |
21,28,53,57 Amp
10/3
Ligation
Tonginsert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
---|---|---|---|---|---|
gBlock | 1 | pRS426(Sma1 cut)/326 | 1 | 2 | 4 |
10/3
Restriction Enzyme Digestion
Tongsample | volume(ul) |
---|---|
gBlock(McHKT2) | 20 |
10×H buffer | 10 |
EcoR1 | 1 |
Pst1 | 1 |
DW | 68 |
Total | 100 |
10/3
PCR
TongMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 8 |
10uM dNTPs | 3.2 |
10uM Z094 | 1 |
10uM Z095 | 1 |
template① | 1 |
template② | 1 |
template③ | 1 |
DW | 23.4 |
PrimeSTAR HS | 0.4 |
Total | 40 |
10/3
PCR
ShimazoeMaterial | Volume |
---|---|
5× Q5 Reaction Buffer | 8 |
10uM dNTPs | 3.2 |
10uM Z080 | 1 |
10uM Z083 | 1 |
template | 1 |
Polymerase | 0.4 |
DW | 25.4 |
Total | 40 |
Material | Volume |
---|---|
5× Q5 Reaction Buffer | 8 |
10uM dNTPs | 3.2 |
10uM Z078 | 1 |
10uM Z083 | 1 |
template | 1 |
Polymerase | 0.4 |
DW | 25.4 |
Total | 40 |
Fgβ-Stalk, SdrG-Stalk
10/4
Nanodrop
Nambusample | 1 | 2 | 9 | 12 | 17 | 20 | 25 | 33 | 40 | 46 |
---|---|---|---|---|---|---|---|---|---|---|
concentration(ng/ul) | 182.6 | 199.9 | 323.0 | 246.3 | 264.2 | 283.5 | 192.9 | 187.5 | 212.5 | 199.8 |
10/4
Restriction Enzyme Digestion
Nambusample | 1/2/9/12/17/20/25/33/40/46 |
---|---|
volume(ul) | 5 |
10×H buffer | 2 |
EcoR1 | 1 |
Pst1 | 1 |
DW | 11 |
Total | 20 |
10/4
Colony PCR
NambuMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 42.5 |
10uM VF2 | 0.85 |
10uM Z018 | 0.85 |
DW | 40.8 |
Total | 85 |
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 3min
72℃ 3min
15℃ ∞
(*× 30cycle)
10/4
Liquid Culture
NambuAtNHXS1 “2” “3”
10/4
Ligation
ShimazoeMaterial | Volume(ul) |
---|---|
Ligation high | 2 |
vector | 1 |
insert | 1 |
Total | 4 |
C1/L | C2/L | C3/L | C4/L | C5/L | C2/H | C3/H | |
---|---|---|---|---|---|---|---|
backbone | pSB1C3 | pRS316 | pRS316 | pSB1C3 | pSB1C3 | YCplac195 | YCplac195 |
insert | McHKT2 | ZrFPS1 | ZrFPS1 | SdrG | Fgβ | ZrFPS1 | ZrFPS1 |
10/4
Restriction Enzyme Digestion
Shimazoematerial | volume(ul) |
---|---|
DNA | 15 |
10×H buffer | 8 |
EcoR1 | 1 |
Pst1 | 1 |
DW | 55 |
Total | 80 |
10/5
Liquid Culture
NambuFgβ 34,35,36,37,38,39
10/5
Colony PCR
NambuMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 87.5 |
10uM m13f | 1.75 |
10uM m13r | 1.75 |
DW | 84.0 |
Total | 175.0 |
Stalk
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 2m30s
72℃ 3min
15℃ ∞
(*× 30cycle)
10/5
Restriction Enzyme Digestion
Shimazoesample | volume(ul) |
---|---|
DNA | 10 |
10×H buffer | 2 |
Sca1 | 1 |
DW | 7 |
Total | 20 |
R | R | R | R | R | R | R | R |
---|---|---|---|---|---|---|---|
Fgβ | Fgβ | Fgβ | Fgβ | SdrG | SdrG | SdrG | SdrG |
pIEG | pIEG | 21 | 28 | pIEG | pIEG | 53 | 57 |
10/5
Transformation
TongLabeling | Yeast type | Gene |
---|---|---|
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) |
・SDS-PAGE
・Western Blotting
10/7
Colony PCR
ShimazoeMaterial | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 120 |
10uM VR | 2.4 |
10uM VF | 2.4 |
DW | 115.2 |
Total | 240 |
Material | Volume(ul) |
---|---|
2× Emerald amp MAX PCR Master Mix | 100 |
10uM m13r | 2 |
10uM m13f | 2 |
DW | 96 |
Total | 200 |
10/10
Transformation
TongLabeling | Yeast type | URA | LEU | HIS |
---|---|---|---|---|
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 |