Difference between revisions of "Team:Kyoto/Notebook"
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<td><a href="#1009">9</a></td> | <td><a href="#1009">9</a></td> | ||
<td><a href="#1010">10</a></td> | <td><a href="#1010">10</a></td> | ||
| − | <td | + | <td>11</td> |
| − | <td | + | <td>12</td> |
| − | <td | + | <td>13</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td | + | <td>14</td> |
| − | <td | + | <td>15<</td> |
| − | <td | + | <td>16</td> |
<td>17</td> | <td>17</td> | ||
<td>18</td> | <td>18</td> | ||
Latest revision as of 13:51, 17 October 2018
| July | ||||||
|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| 15 | 16 | 17 | 18 | 19 | 20 | 21 |
| 22 | 23 | 24 | 25 | 26 | 27 | 28 |
| 29 | 30 | 31 | ||||
| August | ||||||
|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | |||
| 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| 12 | 13 | 14 | 15 | 16 | 17 | 18 |
| 19 | 20 | 21 | 22 | 23 | 24 | 25 |
| 26 | 27 | 28 | 29 | 30 | 31 | |
| September | ||||||
|---|---|---|---|---|---|---|
| 1 | ||||||
| 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| 16 | 17 | 18 | 19 | 20 | 21 | 22 |
| 23 | 24 | 25 | 26 | 27 | 28 | 29 |
| 30 | ||||||
| October | ||||||
|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | |
| 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 14 | 15< | 16 | 17 | 18 | 19 | 20 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | 31 | |||
7/3
Restriction Enzyme Digestion
Tong, Tamukai| component | volume(μl) |
|---|---|
| BSA | 0.5 |
| buffer(10xH) | 5 |
| EcoR1 | 0.5 |
| Pst1 | 0.5 |
| Dpn1 | 0.5 |
| dH2O | 18 |
add 4μl linearized plasmid backbone (25ng/μl)
add 4μl Enzyme Master Mix
digest 37℃, 30min
add 8μl NaOAc (pH5.2,3M)
add 16μl 99.5% EtOh
Vortex 2sec
add 184μl 99.5% EtOH
add 72μl DW
7/5
OE-PCR
Nambu, Shimazoe| sample | Primer Forward | volume (ul) | Primer Reverse | volume (ul) | template DNA | volume (ul) | Q5 HF 2xMasterMix (ul) | DW (ul) |
|---|---|---|---|---|---|---|---|---|
| 1 | Z007 | 1.25 | Z009 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
| 2 | Z008 | 1.25 | Z011 | 1.25 | GFP | 1.0 | 1.0 | 9.0 |
| 3 | Z010 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
| 4 | Z007 | 1.25 | Z014 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
| 5 | Z013 | 1.25 | Z016 | 1.25 | RFP | 1.0 | 1.0 | 9.0 |
| 6 | Z015 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
7/6
OE-PCR
Nambu, Shimazoe| sample | Primer Forward | volume (ul) | Primer Reverse | volume (ul) | Template DNA | volume (ul) | Q5 HF2x Master Mix (ul) | DW (ul) |
|---|---|---|---|---|---|---|---|---|
| 1 | Z007 | 1.25 | Z009 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
| 2 | Z010 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
| 3 | Z007 | 1.25 | Z014 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
| 4 | Z015 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
| 5* | Z007 | 1.25 | Z009 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
| 6* | Z010 | 1.25 | Z012 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
| 7* | Z007 | 1.25 | Z014 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
| 8* | Z015 | 1.25 | Z012 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
*sample5~8 x 2set
7/6
colony_PCR
| Mangrin colony | DW (ul) | Q5 HF2x MasterMix (ul) | m13 Forward (ul) | m13 Reverse (ul) |
|---|---|---|---|---|
| 1 | 2.0 | 2.5 | 0.25 | 0.25 |
| 2 | 2.0 | 2.5 | 0.25 | 0.25 |
| 3 | 2.0 | 2.5 | 0.25 | 0.25 |
| 4 | 2.0 | 2.5 | 0.25 | 0.25 |
| 5 | 2.0 | 2.5 | 0.25 | 0.25 |
Store PCR product at 4℃.
7/10
Restriction Enzyme Digestion
Tong, Tamukai| component | volume(μl) |
|---|---|
| BSA | 0.5 |
| buffer(10xH) | 5 |
| EcoR1 | 0.5 |
| Pst1 | 0.5 |
| Dpn1 | 0.5 |
| dH2O | 18 |
add 4, 8 μl (add two by two to sample)
add 4, 8μl Enzyme master mix (add two by two to sample)
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
Nambu| sample | KOD one PCR master mix (ul) | TDH promoter | volume (ul) | ORF | volume (ul) | CYC terminator | volume (ul) | DW (ul) |
|---|---|---|---|---|---|---|---|---|
| A | 12.5 | 5 | 1.5 | GFP | 1.5 | 6 | 1.5 | 8.0 |
| B | 12.5 | 7 | 1.5 | RFP | 1.5 | 8 | 1.5 | 8.0 |
| C | 12.5 | 5 | 1.5 | GFP | 1.5 | 6 | 1.5 | 8.0 |
| D | 12.5 | 7 | 1.5 | RFP | 1.5 | 8 | 1.5 | 8.0 |
PCR
98℃ 0:30
98℃* 0:10
45℃* 0:05
68℃* 0:05
68℃ 2:00
10℃
(* x 30 cycle)
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
Tong| Component | (1L) | (900mL) | (600mL) |
|---|---|---|---|
| Peptone | 20g | 18g | 12g |
| Yeast Extract | 10g | 9g | 6g |
| Agar | 15g | 13.5g | 9g |
| DW | xmL | 810ml | 480mL |
| Glucose+DW | 20g+ymL (x+y=1000) | 18g | 12g |
fluid culture
cultured 9 yeast strains in liquid YPD.
Put 15 ml of YPD medium in a 50 ml sterile tube
push the yeast with a white chip, put it in the tube
loosen the lid and put it in the shaker
For each 8 samples, transferred to a 1.5 ml tube 1mL each.
For soy sauce yeast only 15 ml was centrifuged, decantation was repeated and scaled down to 1 ml.
8/13
Ligation
Nambu| Number | 1 | 2 | 3 | 4 | 5 | 6 |
|---|---|---|---|---|---|---|
| vector | 426 | 316 | 426 | 316 | 426 | 316 |
| insert | Mangrin | Mangrin | ZrFPS1-2 | ZrFPS1-2 | McHKT2 | McHKT2 |
Mix the following material (Contamination are shown in the table above)
Plasmid(vector): 0.5µL
Insert: 1.0µl
Ligation High: 1.5µl
Put in Heatblock
8/13
YPD culture
Tong| NaCl 25%(400mL) | null |
|---|---|
| Tryptone | 8g |
| Yeast Extract | 4g |
| Agar | 8g |
| Glucose | 8g |
| NaCl | 100g |
| Normal | null |
|---|---|
| Tryptone | 4g |
| Yeast Extract | 2g |
| Agar | 4g |
| Glucose | 4g |
Make
25% NaCl YPD solid medium 400 mL
Normal YPD solid medium 200 mL
YPD liquid medium 500 mL
8/14
Transformation
Tamukai| Sample Name | Sample Volume (μl) | Competent Cells/(μl) |
|---|---|---|
| Mangrin | 3 | 15 |
| ZrFPS1-2 | 3 | 15 |
| McHKT2 | 3 | 15 |
PCR Colony
98℃ 0:30 number Backbone Insert
98℃* 0:10 301~306 SmaCIP AtHKT1
57℃* 0:30 307~317 SmaCIP SseNHX1
72℃* 1:00 318~326 SmaCIP ZrFPs1-1st
72℃ 2:00 327~337 SmaCIP AtNHXS1
4℃ ∞ 338~356 SmaCIP FgβF3
(*× 30 cycle)
8/18
Liquid Culture
Nambu| Material | Volume |
|---|---|
| LB | 90ml |
| Amp | 90ul |
mix materials ( Finally concentration Amp 100 ug/ml )
put colony* into the liquid
(*colony number: 304,309,310,313,314,315,317,322,325,336,342,343,352,353,354)
put in incubator at 37℃ for 18 hours
8/18
Liquid Culture
Tong| Number | Material |
|---|---|
| 547 | WT |
| 554 | NHA1Δ |
| 555 | G19, ΔENA1-4 |
| 557 | NHA1Δ, ENA1Δ |
| 559 | ENA1Δ |
| 561 | Z.roxii ATCC42981 |
| 562 | Z.roxii CBS732 |
| 564 | Z.roxii NBRC1876 |
37℃, 130rpm
prepared each two pieces of plates of 0,5,10,15,20,25% salt concentration and observed the yeasts.
mix the following material
SmaCIP (Backbone) : 1ul
gBlock (Insert) : 1ul
Ligation High : 2ul
(Total : 4ul)
putin HeatBlock overnight at 18℃
8/19
PCR
Nambu| sample | content (gBlock) | primer1 | primer2 |
|---|---|---|---|
| 1 | Mangrin | Z026 | Z027 |
| 2 | ZrFPS1_2nd | Z026 | Z027 |
| 3 | McHKT2 | Z026 | Z027 |
| 4 | AtHKT1 | Z026 | Z027 |
| 5 | ZrFPS1_1st | Z026 | Z027 |
| 6 | AtNHXS1 | Z026 | Z027 |
| 7 | SseNHX1 | Z026 | Z027 |
| 8 | AVP1_1st + AVP1_2nd | Z026 | Z027 |
94℃ 2:00
98℃* 0:10
48℃ *0:30
68℃ *3:00
68℃ 0:10
10℃
* x 30 cycle
8/20
Ligation
Nambu| number | gBlock (Insert) | volume (ul) | SmaCIP (backbone) (ul) | Ligation High (ul) |
|---|---|---|---|---|
| 7 | SdrG | 1 | 1 | 2 |
| 8 | SdrG-Fg overlap | 1 | 1 | 2 |
| 9 | ZrGPD1 | 1 | 1 | 2 |
Put in Incubator overnight at 18℃.
8/20
Transformation
Nambu| Sample Name | Sample Volume (μl) | Competent Cells/(μl) | Medium |
|---|---|---|---|
| Mangrin | 4 | 16 | SOC Medium |
| ZrFPS1_2nd | 4 | 16 | SOC Medium |
| McHKT2 | 4 | 16 | SOC Medium |
| AtHKT1 | 4 | 16 | SOC Medium |
| ZrFPS1_1st | 4 | 16 | SOC Medium |
| AtNHXS1 | 4 | 16 | SOC Medium |
8/21
Restriction Enzyme Digestion
Shimazoe| plasmid | plasmid volume (ul) | DW (ul) | 10x Fast digest buffer (ul) | EcoR1 (ul) | Pst1 (ul) | Total (ul) |
|---|---|---|---|---|---|---|
| 17 | 2 | 14 | 2 | 1 | 1 | 20 |
| 19 | 1 | 14 | 2 | 1 | 1 | 20 |
| 26 | 2 | 14 | 2 | 1 | 1 | 20 |
| 110 | 2 | 14 | 2 | 1 | 1 | 20 |
| 120 | 2 | 14 | 2 | 1 | 1 | 20 |
8/21
Digest plasmid
Tong| plasmid | plasmid volume (ul) | DW (ul) | 10x Fast Digest Buffer (ul) | EcoR1 (ul) | Pst1 (ul) |
|---|---|---|---|---|---|
| 304 | 2 | 14 | 2 | 1 | 1 |
| 309 | 2 | 14 | 2 | 1 | 1 |
| 313 | 2 | 14 | 2 | 1 | 1 |
| 314 | 2 | 14 | 2 | 1 | 1 |
| 317 | 2 | 14 | 2 | 1 | 1 |
| 325 | 2 | 14 | 2 | 1 | 1 |
| 342 | 2 | 14 | 2 | 1 | 1 |
| 322 | 2 | 14 | 2 | 1 | 1 |
| 336 | 2 | 14 | 2 | 1 | 1 |
| 343 | 2 | 14 | 2 | 1 | 1 |
| 352 | 2 | 14 | 2 | 1 | 1 |
| 353 | 2 | 14 | 2 | 1 | 1 |
| 354 | 2 | 14 | 2 | 1 | 1 |
15min 37℃ incubate
8/25
OE-PCR
Shimazoe| plasmid | plasmid volume (ul) | DW (ul) | Q5 (ul) | primer1 | primer1 volume (ul) | primer2 | primer2 volume (ul) |
|---|---|---|---|---|---|---|---|
| 38 (AVP1_1st) | 0.5 | 3.5 | 5 | Z064 | 0.5 | Z026(p) | 0.5 |
| 83 (AVP1_2nd) | 0.5 | 3.5 | 5 | Z065 | 0.5 | Z027(p) | 0.5 |
8/25
colony_PCR
Shimazoe| sample | Q5 (ul) | DW (ul) | primer m13f (ul) | primer m13r (ul) |
|---|---|---|---|---|
| 38 | 2.5 | 2.0 | 0.25 | 0.25 |
| 83 | 2.5 | 2.0 | 0.25 | 0.25 |
colony PCR x 2 set
8/26
Liquid Culture
Nambu| sample | LB (ml) | Amp(100mg/ml) (ul) |
|---|---|---|
| 505 | 50 | 50 |
| 506 | 50 | 50 |
| 507 | 50 | 50 |
| 508 | 50 | 50 |
| 516 | 50 | 50 |
| 522 | 50 | 50 |
| 530 | 50 | 50 |
Put them in incubator at 37℃ for 19 hours.
8/27
Miniprep_Nanodrop
Nambusample:505,506,507,508,516,522,530
98℃* 10s
50℃* 5s
68℃* 20s
68℃ 1min
4℃ ∞
(*× 40 cycle)
8/28
PCR
Shimazoe| Component | Amount(μl) |
|---|---|
| DW | 8.7 |
| KOD | 10 |
| Z027℗ | 0.6 |
| Z065 | 0.6 |
| 83(template) | 0.1 |
| Total | 20 |
PCR
98℃* 10s
50℃* 5s
68℃* 20s
68℃ 1min
4℃ ∞
(*× 40 cycle)
8/28
OE-PCR
Shimazoe| Component | Amount(μl) |
|---|---|
| DW | 20 |
| Z026℗ | 1.5 |
| Z027℗ | 1.5 |
| KOD one | 25 |
| AVP1-1st | 1 |
| AVP1-2nd | 1 |
| Total | 50 |
PCR
98℃* 10s
65℃* 5s
68℃* 30s
68℃ 1min
4℃ ∞
(*× 25 cycle )
8/28
Colony PCR
Shimazoe| Component | Amount(μl) |
|---|---|
| 2× Gflex PCR Buffer | 162.5 |
| primer m13f | 6.5 |
| primer m13r | 6.5 |
| Tks Gflex DNA polymerase | 6.5 |
| DW | 143 |
| Colony | Poke |
| Total | 325 |
PCR
number
94℃ 1min SdrG 601~611
98℃* 10s Sdr-Fg 612~623
55℃* 15s ZrGPD1 624~631
68℃* 2min Mangrin 633~638
4℃ ∞ Zr-FPS1-2nd 639~642
(*× 30 cycle) McHKT2 643~649
ZrFPS1-1st 650~657
8/28
Liquid Culture
Tong| Number | Material |
|---|---|
| 547 | WT |
| 554 | NHA1Δ |
| 555 | G19, ΔENA1-4 |
| 557 | NHA1Δ, ENA1Δ |
| 559 | ENA1Δ |
| 561 | Z.roxii ATCC42981 |
| 562 | Z.roxii CBS732 |
| 564 | Z.roxii NBRC1876 |
8/28
YPD culture, LB culture
Tong| 100ml 9% YPD | 50ml YPD | 250ml LB | |
|---|---|---|---|
| Peptone(g) | 2 | 1 | 2.5 |
| Yeast Extract(g) | 1 | 0.5 | 1.25 |
| Agar(g) | 1.5 | 0.75 | 3.75 |
| DW(ml) | 100 | 50 | 250 |
| Glucose(g) | 2 | 1 | × |
| NaCl(g) | 9 | × | 1.25 |
8/29
PCR
Shimazoe| Component | Amount(μl) |
|---|---|
| 5× Q5 Reaction Buffer | 15 |
| 10mM dNTPs | 1.5 |
| 10uM Z041 | 3.75 |
| 10uM Z042 | 3.75 |
| Template | Poke |
| Q5 DNA Polymerase | 0.75 |
| DW | 47.25 |
| Total | 72 |
Template
AtHKT1(304), SseNHXS1(317), AtNHXS1(336)
PCR
98℃ 30s
98℃* 10s
65℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)
8/29
PCR
Shimazoe| Component | Amount(μl) |
|---|---|
| 5× Q5 Reaction Buffer | 5 |
| 10mM dNTPs | 0.5 |
| 10uM Z041 | 1.25 |
| 10uM Z042 | 1.25 |
| Template | Poke |
| Q5 DNA Polymerase | 0.25 |
| DW | 16.75 |
| Total | 24 |
Template
FgβF3
PCR
98℃ 30s
98℃* 10s
65℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)
8/30
Reaction Enzyme Digestion
Shimazoe| plasmid | plasmid(ul) | 10× T(ul) | 10× BSA(ul) | SmaⅠ(ul) | CIAP(ul) | DW(ul) | Total/(ul) |
|---|---|---|---|---|---|---|---|
| 426 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
| 316 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
| 425 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
| 315 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
| 423 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
| 313 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
6 sample
37℃ overnight
8/30
OE-PCR
Shimazoe| sample | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | Z026-Z039 (ul) | Z040-Z027 (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| A | AtHKT1 | 0.5 | 2 | 0.2 | 0.5 | 0.5 | 0.5 | 0.5 | 0.1 | 5.2 | 10 |
| B | SseNHX1 | 0.5 | 2 | 0.2 | 0.5 | 0.5 | 0.5 | 0.5 | 0.1 | 5.2 | 10 |
| C | AtNHXS1 | 0.5 | 2 | 0.2 | 0.5 | 0.5 | 0.5 | 0.5 | 0.1 | 5.2 | 10 |
PCR
98℃ 30s
98℃* 10s
72℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 25 cycle)
8/30
PCR
Shimazoe| template | volume (ul) | primer① | volume (ul) | primer② | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|---|---|---|
| AVP1-1st | 0.5 | 10uM Z026℗ | 0.5 | 10uM Z064 | 0.5 | 2 | 0.2 | 0.1 | 6.2 | 10 |
| AVP1-2nd | 0.5 | 10uM Z027℗ | 0.5 | 10uM Z065 | 0.5 | 2 | 0.2 | 0.1 | 6.2 | 10 |
PCR
98℃ 30s
98℃* 10s
67℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 25 cycle)
8/30
OE-PCR
Shimazoe| number | template | volume (ul) | 10uM Z026℗ | 10uM Z027℗ | Z026-Z039 (ul) | Z040-Z027 (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|---|---|---|---|
| A | AtHKT1 | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
| B | SseNHX1 | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
| C | AtNHXS1 | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
| D | GFP | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
| E | RFP | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
PCR
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
8/30
PCR
Shimazoe| template | volume (ul) | 10uM primer① | volume (ul) | 10uM primer② | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|---|---|---|
| AVP1-1st (PCR products) | 1 | Z026℗ | 1.25 | Z064 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| AVP1-1st (Plasmid) | 1 | Z026℗ | 1.25 | Z064 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| AVP1-2nd (PCR products) | 1 | Z027℗ | 1.25 | Z065 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| AVP1-2nd (Plasmid) | 1 | Z027℗ | 1.25 | Z065 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
PCR
98℃ 30s
98℃* 10s
65℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)
・add 3ul of cultures to 3ml of nutrient mediums
・measure OD
・concussion culture (30%, 180rpm)
8/31
OE-PCR
| sample | template | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | 10uM Z026℗ | 10uM Z027℗ | Z026-Z039 (ul) | Z040-Z027 (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|---|---|---|---|
| A | AtHKT1 | 5 | 10 | 1 | 2.5 | 2.5 | 5 | 5 | 0.5 | 18.5 | 50 |
| B | AtNHXS1 | 5 | 10 | 1 | 2.5 | 2.5 | 5 | 5 | 0.5 | 18.5 | 50 |
| C | GFP | 5 | 10 | 1 | 2.5 | 2.5 | 5 | 5 | 0.5 | 18.5 | 50 |
| D | RFP | 5 | 10 | 1 | 2.5 | 2.5 | 5 | 5 | 0.5 | 18.5 | 50 |
PCR
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
8/31
PCR
| template | volume (ul) | 10uM primer① | volume (ul) | 10uM primer② | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | AVP1-2nd (PCR products) | 1 | Z065 | 1.25 | Z027℗ | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| 2 | AVP1-2nd (Plasmid) | 1 | Z065 | 1.25 | Z027℗ | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| 3 | GFP (PCR2) | 1 | Z026℗ | 1.25 | Z027℗ | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| 4 | RFP (PCR5) | 1 | Z026℗ | 1.25 | Z027℗ | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
PCR
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)
8/31
OE-PCR
Shimazoe| sample | template | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | Z026-Z039 (ul) | Z040-Z027 (ul) | KOD one (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|---|---|
| A | SseNHX1 | 1 | 0.3 | 0.3 | 1 | 1 | 5 | 1.4 | 10 |
| B | GFP | 1 | 0.3 | 0.3 | 1 | 1 | 5 | 1.4 | 10 |
| C | RFP | 1 | 0.3 | 0.3 | 1 | 1 | 5 | 1.4 | 10 |
PCR
98℃* 10s
50℃* 5s
68℃* 30s
68℃ 30s
(*× 30 cycle)
9/1
Ligation
Shimazoe| Number | 1 | 2 | 3 | 4 |
|---|---|---|---|---|
| vector | pRS 426 | pRS 316 | pRS 423 | pRS 313 |
| insert | AtHKT1 | AtHKT1 | AtNHXS1 | AtNHXS1 |
Mix the following material (Contamination are shown in the table above)
Plasmid(vector): 1.0ul
Insert: 1.0ul
Ligation High: 2.0ul
Put in Heatblock
9/1
OE-PCR
Shimazoe| template | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | Z026-Z039 (ul) | Z040-Z027 (ul) | Q5 (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|---|
| SseNHX1 | 5 | 1.5 | 1.5 | 5 | 5 | 25 | 7 | 50 |
PCR
98℃ 30s
98℃* 10s
50℃* 20s
72℃* 1min30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
9/1
OE-PCR
Shimazoe| template | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|---|
| AVP1-1st | 1 | 1.25 | 1.25 | 5 | 0.5 | 0.25 | 14.75 | 25 |
| AVP1-2nd | 1 | 1.25 | 1.25 | 5 | 0.5 | 0.25 | 14.75 | 25 |
PCR
98℃ 30s
98℃* 10s
55℃* 20s
72℃* 2min
72℃ 2min
(*× 30 cycle)
9/1
Colony PCR
Shimazoe| Material | Volume |
|---|---|
| 2× Emerald amp MAX PCR Master Mix | 375 |
| 10uM m13f | 7.5 |
| 10uM m13r | 7.5 |
| DW | 360 |
| Total | 750 |
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 4min
72℃ 4min
4℃ ∞
(*× 30cycle)
on ice 20min
98℃ 10s
98℃* 10s
50℃* 5s
68℃* 30s
68℃ 30s
(*× 30cycle)
9/2
PCR
Shimazoe| Material | Volume |
|---|---|
| 5× Q5 Reaction Buffer | 5 |
| 10uM dNTPs | 0.5 |
| 10uM primer① | 1.25 |
| 10uM primer② | 1.25 |
| template | 1 |
| Q5 DNA Polymerase | 0.25 |
| DW | 15.75 |
| Total | 25 |
primer① (Z026/Z040)
primer② (Z039/Z027)
98℃ 30s
98℃* 10s
54℃* 20s
72℃* 30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
9/2
OE-PCR
Shimazoe| Material | Volume |
|---|---|
| 2× Emerald Amp MAX | 12.25 |
| 10uM Z026℗ | 0.25 |
| 10uM Z027℗ | 0.25 |
| AVP1-1st | 1 |
| AVP1-2nd | 1 |
| DW | 10.25 |
| Total | 25 |
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 3m30s
72℃ 3m30s
4℃ ∞
(*× 30 cycle)
9/2
OE-PCR
Shimazoe| Material | Volume |
|---|---|
| 5× Q5 Reaction Buffer | 10 |
| 10uM Z026℗ | 2.5 |
| 10uM Z027℗ | 2.5 |
| 10uM dNTPs | 1 |
| template⓪ | 5 |
| template① | 5 |
| template② | 5 |
| Q5 DNA Polymerase | 0.5 |
| DW | 18.5 |
| Total | 50 |
template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
9/3
Ligation
Tong| sample | volume(ul) | pRS316(ul) | Ligation High(ul) | Total(ul) |
|---|---|---|---|---|
| Mangrin | 1 | 2 | 3 | 6 |
| ZrFPS1-1st | 1 | 2 | 3 | 6 |
| ZrFPS1-2nd | 1 | 2 | 3 | 6 |
| ZrGPD1 | 1 | 2 | 3 | 6 |
| McHKT2 | 1 | 2 | 3 | 6 |
| SdrG | 1 | 2 | 3 | 6 |
| SdrG-Fgβ | 1 | 2 | 3 | 6 |
18℃overnight
on ice 20min
42℃ 45sec
on ice 3min
SOC 550
37℃ 40min
9/4
PCR
Tong| Material | volume (ul) |
|---|---|
| DW | 22.8 |
| Template (pIEG-SSS) | 1.6 |
| Primer F (Z067) | 2 |
| Primer R (Z066) | 2 |
| DNA Pol (Q5) | 0.4 |
| Reaction buffer | 8 |
| dNTP mix | 3.2 |
| Total | 40 |
98℃ 30s
98℃* 10s
48℃* 30s
72℃* 3min
72℃ 2min
4℃ ∞
(*× 30cycle)
9/4
OE-PCR
Shimazoe| Material | Volume |
|---|---|
| 5× Q5 Reaction Buffer | 10 |
| 10uM Z026℗ | 2.5 |
| 10uM Z027℗ | 2.5 |
| 10uM dNTPs | 1 |
| template⓪ | 5 |
| template① | 5 |
| template② | 5 |
| Q5 DNA Polymerase | 0.5 |
| DW | 18.5 |
| Total | 50 |
template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
9/4
OE-PCR
Shimazoe| Material | Volume |
|---|---|
| 5× Q5 Reaction Buffer | 10 |
| 10uM Z026 | 2.5 |
| 10uM Z027 | 2.5 |
| 10uM dNTPs | 1 |
| template⓪ | 5 |
| template① | 5 |
| template② | 5 |
| Q5 DNA Polymerase | 0.5 |
| DW | 18.5 |
| Total | 50 |
template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
9/4
Colony PCR
Shimazoe| Material | Volume(ul) |
|---|---|
| 2× Emerald amp MAX PCR Master Mix | 40 |
| 10uM m13f | 0.8 |
| 10uM m13r | 0.8 |
| DW | 38.4 |
| Total | 80 |
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 4min
72℃ 4min
4℃ ∞
(*× 30cycle)
9/4
Ligation
Tong| number | sample | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
|---|---|---|---|---|---|---|
| 1 | AtHKT1 | 1 | 316,426 | 2 | 3 | 6 |
| 2 | AtNHXS1 | 1 | 423,313 | 2 | 3 | 6 |
| 3 | SseNHX1 | 1 | 423,313 | 2 | 3 | 6 |
9/5
PCR
Tong| Material | volume (ul) |
|---|---|
| DW | 22 |
| Template (pIEG-SSS) | 1 |
| Primer F (Z067) | 1 |
| Primer R (Z066) | 1 |
| PrimeSTAR Max | 25 |
| Total | 50 |
98℃* 10s
55℃* 15s
72℃* 35s
(*× 30cycle)
9/5
OE-PCR
Shimazoe| Material | Volume |
|---|---|
| 5× Q5 Reaction Buffer | 12 |
| 10uM Z026℗ | 3 |
| 10uM Z027℗ | 3 |
| 10uM dNTPs | 1.2 |
| template⓪ | 6 |
| template① | 6 |
| template② | 6 |
| Q5 DNA Polymerase | 0.6 |
| DW | 22.2 |
| Total | 60 |
template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
9/5
Transformation
Shimazoe| 1 | 2 | 3 | 4 | 5 | 6 | |
|---|---|---|---|---|---|---|
| sample | AtHKT1 | AtHKT1 | AtNHXS1 | AtNHXS1 | SseNHX1 | SseNHX1 |
| vector | 426 | 316 | 423 | 313 | 423 | 313 |
| competent cell (ul) | 20 | 30 | 20 | 30 | 20 | 30 |
9/5
PCR
Shimazoe| template | volume (ul) | 10uM primer① (ul) | volume (ul) | 10uM primer② (ul) | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|---|---|---|
| AVP1-1st | 1 | Z026 | 1.25 | Z071 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| AVP1-2nd | 1 | Z027 | 1.25 | Z072 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| AVP1-1st | 1 | Z026 | 1.25 | Z070 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)
9/5
OE-PCR
Shimazoe| Material | Volume |
|---|---|
| 5× Q5 Reaction Buffer | 10 |
| 10uM Z026℗ | 2.5 |
| 10uM Z027℗ | 2.5 |
| AVP1-1st | 2 |
| AVP1-2nd | 2 |
| 10uM dNTPs | 1 |
| Q5 DNA Polymerase | 0.5 |
| DW | 24.5 |
| Total | 50 |
template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
9/5
Colony PCR
Tamukai| Material | Volume(ul) |
|---|---|
| 2× Emerald amp MAX PCR Master Mix | 180 |
| 10uM m13f | 7.2 |
| 10uM m13r | 7.2 |
| DW | 180 |
| Total | 373.4 |
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 4min
72℃ 4min
4℃ ∞
(*× 30cycle)
・Seperate into 17 microtube 150uL each
・incubate in 70℃ for 10min
・on ice for 5min
・Add Precipation Reagent 90ul, vortex, on ice 10min, remove supreme
・Add 80ul Phend-chloroform, vortex, max rpm (15,000rpm) 10min, remove supreme
・ -30℃
9/5
OE-PCR
Shimazoe| Material | Volume(ul) |
|---|---|
| 2× Gflex PCR Buffer | 25 |
| 10uM Z026℗ | 1 |
| 10uM Z027℗ | 1 |
| template⓪ | 1 |
| template① | 1 |
| template② | 1 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 19 |
| Total | 50 |
⓪(SseNHX1/RFP)
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
(*× 30 cycle)
9/5
Ligation
Shimazoe| insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
|---|---|---|---|---|---|
| AtHKT1 | 2 | 316 | 2 | 4 | 8 |
| AtHKT1 | 2 | 426 | 2 | 4 | 8 |
| AtNHXS1 | 2 | 423 | 2 | 4 | 8 |
| AtNHXS1 | 2 | 313 | 2 | 4 | 8 |
| SseNHX1 | 2 | 423 | 2 | 4 | 8 |
| SseNHX1 | 2 | 313 | 2 | 4 | 8 |
9/6
Colony PCR
Shimazoe| Material | Volume(ul) |
|---|---|
| 2× Emerald amp MAX PCR Master Mix | 125 |
| 10uM m13f | 2.5 |
| 10uM m13r | 2.5 |
| DW | 120 |
| Total | 250 |
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 2min
72℃ 2min
4℃ ∞
(*× 30cycle)
9/6
OE-PCR
Shimazoe| Material | Volume(ul) |
|---|---|
| 2× Gflex PCR Buffer | 25 |
| 10uM Z026℗ | 1 |
| 10uM Z027℗ | 1 |
| AVP1-1st | 1 |
| AVP1-2nd | 1 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 20 |
| Total | 50 |
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 2min
68℃ 2min
(*× 30 cycle)
9/6
PCR
Shimazoe| Material | Volume |
|---|---|
| 5× Q5 Reaction Buffer | 5 |
| 10uM primer① | 1.25 |
| 10uM primer② | 1.25 |
| 10uM dNTPs | 0.5 |
| template | 1 |
| Q5 DNA Polymerase | 0.25 |
| DW | 15.75 |
| Total | 25 |
ZrGPD1(primer①②:Z041,Z042 template:506), SdrG(primer①②:Z044,Z045 template:752cp)
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)
9/7
Restriction Enzyme Digestion
Tong| plasmid | plasmid(ul) | 10×K buffer(ul) | 10×H buffer(ul) | 10× BSA(ul) | EcoRⅠ(ul) | NcoⅠ(ul) | PstⅠ(ul) | DpnⅠ(ul) | DW(ul) | Total/(ul) |
|---|---|---|---|---|---|---|---|---|---|---|
| 38 | 10 | 4 | × | 4 | 2 | 2 | × | × | 28 | 40 |
| 83 | 10 | 4 | × | 4 | × | 2 | 2 | × | 28 | 40 |
| pSB1C3 | 20 | × | 4 | × | 2 | × | 2 | 2 | 10 | 40 |
| YCplac111 | 2 | × | 4 | × | 2 | × | 2 | × | 30 | 40 |
9/8
Restriction Enzyme Digestion
Tong| volume(ul) | 10×H buffer(ul) | EcoRⅠ(ul) | PstⅠ(ul) | DW(ul) | Total/(ul) | |
|---|---|---|---|---|---|---|
| DNA 404 | 5 | 4 | 2 | 2 | 27 | 40 |
9/8
Ligation
Tong| sample | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
|---|---|---|---|---|---|
| AtHKT1 | 2 | 316 | 2 | 4 | 8 |
| AtHKT1 | 2 | 426 | 2 | 4 | 8 |
| AtNHXS1 | 2 | 423 | 2 | 4 | 8 |
| AtNHXS1 | 2 | 313 | 2 | 4 | 8 |
| SseNHX1 | 2 | 423 | 2 | 4 | 8 |
| SseNHX1 | 2 | 313 | 2 | 4 | 8 |
9/8
OE-PCR
Tong| template | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | promoter(ul) | terminator(ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|---|---|---|
| ZrGPD1 ORF | 5 | 2.5 | 2.5 | 10 | 1 | 5 | 5 | 0.5 | 18.5 | 50 |
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1min30s
72℃ 2min
4℃ ∞
(*× 30cycle)
・on ice 10min
・42℃ 45sec
・+SOC 300ul
・37℃ 40min
9/8
OE-PCR
Tong| template① | volume (ul) | template② | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | 2× Prime Star (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|---|
| AVP1-1st | 0.5 | AVP1-2nd | 0.5 | 1 | 1 | 10 | 7 | 20 |
98℃* 10s
55℃* 15s
72℃* 20s
(*× 30cycle)
9/8
PCR
Tong| Material | volume (ul) |
|---|---|
| DW | 22 |
| Template (pIEG-SSS) | 1 |
| Primer F (Z067) | 1 |
| Primer R (Z066) | 1 |
| PrimeSTAR Max | 25 |
| Total | 50 |
98℃* 10s
55℃* 15s
72℃* 35s
(*× 30cycle)
9/8
Restriction Enzyme Digestion
Tong| volume(ul) | 10×M buffer(ul) | EcoRⅠ(ul) | XbaⅠ(ul) | DW(ul) | Total/(ul) | |
|---|---|---|---|---|---|---|
| AVP1-1st (38) | 20 | 4 | 2 | 2 | 12 | 40 |
| YCplac111 | 2 | 4 | 2 | 2 | 30 | 40 |
9/9
OE-PCR
Tong| template | volume (ul) | Stalk (ul) | Z044 (ul) | Z047 (ul) | 2× Prime Star (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|
| SdrG, Fgβ | 1 | 1 | 1 | 1 | 20 | 16 | 40 |
98℃* 10s
55℃* 5s
72℃* 20s
(*× 30cycle)
9/9
OE-PCR
Tong| template | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | promoter(ul) | terminator(ul) | 2× Prime Star (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|---|
| ZrGPD1 | 0.5 | 1 | 1 | 0.5 | 0.5 | 10 | 6.5 | 20 |
9/9
OE-PCR
Tong| template① | volume (ul) | template② | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | 2× Prime Star (ul) | DW (ul) | Total (ul) |
|---|---|---|---|---|---|---|---|---|
| AVP1-1st | 0.5 | AVP1-2nd | 0.5 | 0.5 | 0.5 | 10 | 8 | 20 |
98℃* 10s
55℃* 5s
72℃* 15s
(*× 30cycle)
on ice 20min
9/10
Colony PCR
Tong| Material | Volume(ul) |
|---|---|
| 2× Emerald amp MAX PCR Master Mix | 70 |
| 10uM m13f | 1.4 |
| 10uM m13r | 1.4 |
| DW | 67.2 |
| Total | 140 |
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 4min
72℃ 4min
4℃ ∞
(*× 30cycle)
9/10
Colony PCR
Tamukai| Material | Volume(ul) |
|---|---|
| 2× Emerald amp MAX PCR Master Mix | 400 |
| 10uM m13f | 8 |
| 10uM m13r | 8 |
| DW | 384 |
| Total | 800 |
98℃ 30s
98℃* 10s
60℃* 30s
72℃* 2min
72℃ 2min
4℃ ∞
(*× 30cycle)
9/10
OE-PCR
Shimazoe| Material | Volume(ul) |
|---|---|
| 2× Gflex PCR Buffer | 25 |
| 10uM Z026℗ | 1 |
| 10uM Z027℗ | 1 |
| template⓪ | 1 |
| template① | 1 |
| template② | 1 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 19 |
| Total | 50 |
ZrGPD1
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 30 cycle)
9/10
Colony PCR
Nambu| Material | Volume(ul) |
|---|---|
| 2× Emerald amp MAX PCR Master Mix | 35 |
| 10uM m13f | 0.6 |
| 10uM m13r | 0.6 |
| DW | 33.6 |
| Total | 70 |
98℃ 30s
98℃* 10s
58℃* 30s
72℃* 4min
72℃ 4min
4℃ ∞
(*× 30cycle)
9/10
Restriction Enzyme Digestion
Shimazoe| material | volume(μl) |
|---|---|
| EcoT14Ⅰ | 25 |
| 10×H buffer | 50 |
| λDNA | 71 |
| DW | 354 |
| Total | 500 |
9/11
Restriction Enzyme Digestion
Nambu| material | volume(μl) |
|---|---|
| Mangrin(pRS425) | 10 |
| BSA | 4 |
| 10×H buffer(10xH) | 4 |
| Not1 | 2 |
| TritonX-100 | 4 |
| DW | 16 |
| Total | 40 |
9/11
Restriction Enzyme Digestion
Nambu| material | volume(μl) |
|---|---|
| AVP1 | 5 |
| BSA | 2 |
| 10×H buffer(10xH) | 2 |
| EcoR1 | 1 |
| Nco1 | 1 |
| Pst1 | 1 |
| DW | 8 |
| Total | 20 |
9/11
OE-PCR
Shimazoe| Material | Volume(ul) |
|---|---|
| 2× Gflex PCR Buffer | 30 |
| 10uM Z026℗ | 1.2 |
| 10uM Z027℗ | 1.2 |
| template⓪ | 1.2 |
| template① | 1.2 |
| template② | 1.2 |
| Tks Gflex DNA Polymerase | 1.2 |
| DW | 22.8 |
| Total | 60 |
ZrGPD1
94℃ 1min
98℃* 10s
55℃* 15s
68℃* 1m30s
68℃ 2min
4℃ ∞
(*× 30 cycle)
9/11
Ligation
Shimazoe| insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
|---|---|---|---|---|---|
| GFP | 1 | 426 | 1 | 2 | 4 |
| GFP | 1 | 316 | 1 | 2 | 4 |
| RFP | 1 | 426 | 1 | 2 | 4 |
| RFP | 1 | 316 | 1 | 2 | 4 |
NHA1
98℃ 2min
98℃* 30s
48℃* 30s
68℃* 3m30s
68℃ 5min
4℃ ∞
(*× 30 cycle)
9/11
PCR
Tamukai| material | volume (ul) |
|---|---|
| DW | 47.8 |
| 5× Q5 Reaction Buffer | 16 |
| 10uM dNTPs | 6.4 |
| Q5 DNA Polymerase | 0.8 |
| 10uM Z005 | 4 |
| 10uM Z028 | 4 |
| template(pFA6a-hphMX) | 1 |
| Total | 80 |
ENA1Δ1,2,5
9/11
PCR
Tamukai| material | volume (ul) |
|---|---|
| DW | 45.8 |
| 5× Q5 Reaction Buffer | 16 |
| 10uM dNTPs | 6.4 |
| Q5 DNA Polymerase | 0.8 |
| 10uM Z005 | 4 |
| 10uM Z028 | 4 |
| template(yeast genome) | 1 |
| Total | 80 |
NHA1
9/11
Ligation
Shimazoe| insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
|---|---|---|---|---|---|
| AtHKT1 | 1 | 316 | 1 | 2 | 4 |
| AtHKT1 | 1 | 426 | 1 | 2 | 4 |
| AtNHXS1 | 1 | 423 | 1 | 2 | 4 |
| AtNHXS1 | 1 | 313 | 1 | 2 | 4 |
| SseNHX1 | 1 | 423 | 1 | 2 | 4 |
| SseNHX1 | 1 | 313 | 1 | 2 | 4 |
9/12
Restriction Enzyme Digestion
Nambu| material | volume(μl) |
|---|---|
| CIAP | 1 |
| pRS426 or pRS425 | 2 |
| BSA | 4 |
| 10×H buffer(10xH) | 4 |
| Not1 | 2 |
| TritonX-100 | 4 |
| DW | 23 |
| Total | 40 |
| material | volume(μl) |
|---|---|
| AVP1(on pSB1C3) | 5 |
| BSA | 2 |
| 10×H buffer(10xH) | 2 |
| Not1 | 1 |
| TritonX-100 | 4 |
| DW | 8 |
| Total | 20 |
| 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
Shimazoe| Material | Volume(ul) |
|---|---|
| Ligation high | 4 |
| pRS425 Not1 CIAP | 2 |
| AVP1 Not1 ③ | 2 |
| Total | 8 |
9/13
OE-PCR
Shimazoe| Material | 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
Shimazoe| Material | 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
Shimazoe| material | 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
Shimazoe| Material | 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
Shimazoe| Material | 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
Nambu| material | 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
Shimazoe| Material | 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
Nambu| sample | 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
Tong| Material | 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
Tong| number | 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
Shimazoe| Material | 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
Nambu| material | 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
Tong| Material | 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
Tong| material | 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
Shimazoe| Material | 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
Tong| material | 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
Nambu| sample | 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
Shimazoe| Material | 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
Shimazoe| material | 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
Shimazoe| Material | 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
Nambu| volume(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
Shimazoe| Material | 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
Tong| Material | 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
Shimazoe| material | 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
Shimazoe| Material | 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
Shimazoe| Ligation products | volume(ul) | Competent Cells/(ul) | Total(ul) |
|---|---|---|---|
| SseNHX1/GPD1 | 10 | 50 | 60 |
| Fgβ/SdrG | 10 | 50 | 60 |
9/19
OE-PCR
Tong| Material | 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
Tong| Material | 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
Tong| material | 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
Shimazoe| Material | 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
Tong| Material | 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
Tong| material | 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
Tong| material | 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
Shimazoe| Material | Volume(ul) |
|---|---|
| Ligation high | 2 |
| vector | 1 |
| insert | 1 |
| Total | 4 |
Mangrin : pRS426
AVP1 : pRS425
9/20
Restriction Enzyme Digestion
Nambu| sample | 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
Shimazoe| Material | Volume(ul) |
|---|---|
| dsDNA buffer | 200 |
| dsDNA reagent | 1 |
| dsDNA solution | 1 |
| Total | 202 |
9/21
Ligation
Shimazoe| Material | Volume(ul) |
|---|---|
| Ligation high | 6 |
| vector 426/316 | 2/2 |
| ZrFPS1-1st | 2 |
| ZrFPS1-2nd | 2 |
| Total | 12 |
9/21
SLiCE
Shimazoe| Material | 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
Shimazoe| material | 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
Shimazoe| material | 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
Tong| yeast | 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
Shimazoe| Material | 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
Shimazoe| Material | 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
Shimazoe| Material | 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
Tong| Material | 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
Tong| Number | 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
Nambu| sample | 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
Nambu| sample | 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
Shimazoe| Material | 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
Tong| Material | 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
Tong| material | 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
Nambu| Material | 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
Shimazoe| Material | 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
Nambu| number | 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
Shimazoe| Material | 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
Nambu| Material | 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
Nambu| sample | 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
Nambu| sample | 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
Shimazoe| Material | 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
Shimazoe| Material | 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
Nambu| sample | Plasmid(AVP1.pSB1C3) |
|---|---|
| volume(ul) | 20 |
| 10×H buffer | 4 |
| EcoR1 | 1 |
| Pst1 | 1 |
| CIAP | 1 |
| DW | 13 |
| Total | 40 |
9/27
Transformation(yeast)
Tong| yeast | 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
Tong| number | 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
Shimazoe| material | 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
Shimazoe| material | 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
Shimazoe| Material | 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
Shimazoe| Material | 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
Nambu| sample | 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
Nambu| Stalk | 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
Nambu| sample | 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
Tong| material | 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
Tong| Material | 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
Tong| number | 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
Shimazoe| Material | 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
Shimazoe| material | 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
Nambu| sample | 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
Nambu| Material | 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
Nambu| insert | 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
Nambu| volume(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
Tong| Material | 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
Tong| insert | 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
Tong| Material | 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
Tong| Material | 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
Tong| Material | 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
Shimazoe| Material | 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
Nambu| sample | 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
Nambu| Material | 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
Tong| Material | 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
Tong| Material | 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
Shimazoe| Material | 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
Shimazoe| Material | 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
Nambu| Material | 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
Nambu| Material | 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
Nambu| Mangrin(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
Tong| 1～14 | 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
Tong| Material | 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
Tong| Material | 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
Tong| sample | 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
Shimazoe| Material | 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
Nambu| sample | 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
Tong| insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
|---|---|---|---|---|---|
| gBlock | 1 | pRS426(Sma1 cut)/326 | 1 | 2 | 4 |
10/3
Restriction Enzyme Digestion
Tong| sample | volume(ul) |
|---|---|
| gBlock(McHKT2) | 20 |
| 10×H buffer | 10 |
| EcoR1 | 1 |
| Pst1 | 1 |
| DW | 68 |
| Total | 100 |
10/3
PCR
Tong| Material | 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
Shimazoe| Material | 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
Nambu| sample | 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
Nambu| sample | 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
Nambu| Material | 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
Shimazoe| Material | 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
Shimazoe| material | 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
Nambu| Material | 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
Shimazoe| sample | 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
Tong| Labeling | 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
Shimazoe| Material | 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
Tong| Labeling | 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 |