Difference between revisions of "Team:Kyoto/Notebook"

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<p class="keln_note">37℃, 130rpm<br>prepared each two pieces of plates of 0,5,10,15,20,25% salt concentration and observed the yeasts.</p>
 
<p class="keln_note">37℃, 130rpm<br>prepared each two pieces of plates of 0,5,10,15,20,25% salt concentration and observed the yeasts.</p>
 +
<img src="http://https://2018.igem.org/File:IGKU_b1_030_0.jpeg">
 +
 
<!------------ Table END ------------>
 
<!------------ Table END ------------>
 
<!-- Table Generated by KELN Author: Nakazato -->
 
<!-- Table Generated by KELN Author: Nakazato -->

Revision as of 06:53, 22 September 2018

Notebook

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

7/3

Restriction Enzyme Digestion

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

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

7/5

OE-PCR

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

7/6

OE-PCR

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

*sample5~8 x 2set

7/6

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

Store PCR product at 4℃.

7/10

Restriction Enzyme Digestion

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

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

7/12

OE-PCR

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

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

7/31

Transformation & Blue-White Selection

 Shimazoe

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

8/1

Colony PCR

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

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

8/7

YPD

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

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

8/8

OD measure

 Tong
AOD
5620.38
5610.40
5640.36
5540.33
5550.38
5470.30
5570.32
5590.39

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

8/13

Ligation

 Nambu
Number123456
vector426316426316426316
insertMangrinMangrinZrFPS1-2ZrFPS1-2McHKT2McHKT2

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

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

Put in Heatblock

8/13

YPD culture

 Tong
NaCl 25%(400mL)null
Tryptone8g
Yeast Extract4g
Agar8g
Glucose8g
NaCl100g

Normalnull
Tryptone4g
Yeast Extract2g
Agar4g
Glucose4g

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

8/14

Transformation

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

8/18

Colony PCR

 Nambu
ComponentAmount(μl)
DW112
Q5140
m13f14
m13r14
ColonyPoke
Total280

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

8/18

Liquid Culture

 Nambu
MaterialVolume
LB90ml
Amp90ul

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

8/18

Liquid Culture

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

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

8/19

Ligation

 Nambu
Number123456
gBlockMangrinZrFPS1-2ndMcHKT2AtHKT1ZrFPS1-1stAtNHXS1

mix the following material

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

putin HeatBlock overnight at 18℃

8/19

PCR

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

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

8/20

Ligation

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

Put in Incubator overnight at 18℃.

8/20

Transformation

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

8/21

Restriction Enzyme Digestion

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

8/21

Digest plasmid

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

15min 37℃ incubate

8/25

OE-PCR

 Shimazoe
plasmid plasmid volume (ul)DW (ul)Q5 (ul)primer1 primer1 volume (ul)primer2 primer2 volume (ul)
38 (AVP1_1st)0.53.55Z0640.5Z026(p)0.5
83 (AVP1_2nd)0.53.55Z0650.5Z027(p)0.5

8/25

colony_PCR

 Shimazoe
sampleQ5 (ul)DW (ul)primer m13f (ul)primer m13r (ul)
382.52.00.250.25
832.52.00.250.25

colony PCR x 2 set

8/26

Liquid Culture

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

Put them in incubator at 37℃ for 19 hours.

8/27

OE-PCR

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

8/27

Miniprep_Nanodrop

 Nambu

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

8/28

PCR

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

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

8/28

PCR

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

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

8/28

OE-PCR

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

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

8/28

Colony PCR

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

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

8/28

Liquid Culture

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

8/28

YPD culture, LB culture

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

8/29

PCR

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

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

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

8/29

PCR

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

Template
FgβF3

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

8/30

Reaction Enzyme Digestion

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

6 sample
37℃ overnight

8/30

OE-PCR

 Shimazoe
samplevolume (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)10uM Z026℗ (ul)10uM Z027℗ (ul)Z026-Z039 (ul)Z040-Z027 (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
AAtHKT10.520.20.50.50.50.50.15.210
BSseNHX10.520.20.50.50.50.50.15.210
CAtNHXS10.520.20.50.50.50.50.15.210

PCR

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

8/30

PCR

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

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

8/30

OE-PCR

 Shimazoe
numbertemplatevolume (ul)10uM Z026℗10uM Z027℗Z026-Z039 (ul)Z040-Z027 (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
AAtHKT110.50.51120.20.13.710
BSseNHX110.50.51120.20.13.710
CAtNHXS110.50.51120.20.13.710
DGFP10.50.51120.20.13.710
ERFP10.50.51120.20.13.710

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

8/30

PCR

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

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

8/30

Yeast Selection

 Tong

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

8/31

OE-PCR
sampletemplatevolume (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)10uM Z026℗10uM Z027℗Z026-Z039 (ul)Z040-Z027 (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
AAtHKT151012.52.5550.518.550
BAtNHXS151012.52.5550.518.550
CGFP51012.52.5550.518.550
DRFP51012.52.5550.518.550

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

8/31

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

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

8/31

OE-PCR

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

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

9/1

Ligation

 Shimazoe
Number1234
vectorpRS 426pRS 316pRS 423pRS 313
insertAtHKT1AtHKT1AtNHXS1AtNHXS1

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

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

Put in Heatblock

9/1

OE-PCR

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

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

9/1

OE-PCR

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

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

9/1

Colony PCR

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

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

9/2

Ligation

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

9/2

Transformation

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

on ice 20min

9/2

PCR

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

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

9/2

PCR

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

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

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

9/2

OE-PCR

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

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

9/2

OE-PCR

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

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

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

9/3

Ligation

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

18℃overnight

9/4

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

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

9/4

PCR

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

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

9/4

OE-PCR

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

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

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

9/4

OE-PCR

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

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

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

9/4

Colony PCR

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

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

9/4

Ligation

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

9/5

PCR

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

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

9/5

OE-PCR

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

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

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

9/5

Transformation

 Shimazoe
123456
sampleAtHKT1AtHKT1AtNHXS1AtNHXS1SseNHX1SseNHX1
vector426316423313423313
competent cell (ul)203020302030

9/5

PCR

 Shimazoe
templatevolume (ul)10uM primer① (ul)volume (ul)10uM primer② (ul)volume (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
AVP1-1st1Z0261.25Z0711.2550.50.2515.7525
AVP1-2nd1Z0271.25Z0721.2550.50.2515.7525
AVP1-1st1Z0261.25Z0701.2550.50.2515.7525

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

9/5

OE-PCR

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

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

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

9/5

Colony PCR

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

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

9/5

Genome extracion

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

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


9/5

OE-PCR

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

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

9/5

Ligation

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

9/6

Transformation

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

9/6

Colony PCR

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

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

9/6

OE-PCR

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

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

9/6

PCR

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

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

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

9/7

Restriction Enzyme Digestion

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

9/8

Restriction Enzyme Digestion

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

9/8

Ligation

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

9/8

OE-PCR

 Tong
templatevolume (ul)10uM Z026℗ (ul)10uM Z027℗ (ul)5× Q5 Reaction Buffer (ul)10uM dNTPs (ul)promoter(ul)terminator(ul)Q5 DNA Polymerase (ul)DW (ul)Total (ul)
ZrGPD1 ORF52.52.5101550.518.550

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

9/8

Transformation

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

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

9/8

OE-PCR

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

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

9/8

PCR

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

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

9/8

Restriction Enzyme Digestion

 Tong
volume(ul)10×M buffer(ul)EcoRⅠ(ul)XbaⅠ(ul)DW(ul)Total/(ul)
AVP1-1st (38)204221240
YCplac11124223040

9/8

Ligation

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

9/9

OE-PCR

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

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

9/9

OE-PCR

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

9/9

OE-PCR

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

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

9/9

Ligation

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

9/10

Transformation

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

on ice 20min

9/10

Colony PCR

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

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

9/10

Colony PCR

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

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

9/10

OE-PCR

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

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

9/10

Colony PCR

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

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

9/10

Restriction Enzyme Digestion

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

9/11

Restriction Enzyme Digestion

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

9/11

Restriction Enzyme Digestion

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

9/11

OE-PCR

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

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

9/11

Ligation

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

9/11

PCR

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

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

9/11

PCR

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

ENA1Δ1,2,5

9/11

PCR

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

NHA1

9/11

Ligation

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

9/12

Restriction Enzyme Digestion

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


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

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

4sample(3,6,7,11)

9/12

Ligation

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

9/13

Transformation

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

9/13

OE-PCR

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

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

9/13

OE-PCR

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

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

9/13

PCR

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

9/13

Colony PCR

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

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

9/13

OE-PCR

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

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

9/14

Ligation

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

9/14

Restriction Enzyme Digestion

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

9/14

Colony PCR

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

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

9/15

OE-PCR

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

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

9/15

OE-PCR

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

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

9/15

OE-PCR

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

9/15

OE-PCR

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

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

9/15

Restriction Enzyme Digestion

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


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


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


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

9/16

PCR

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

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

9/16

PCR

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

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

9/16

PCR

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

Stalk

9/16

PCR

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

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

9/16

OE-PCR

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

9/17

OE-PCR

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

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

9/17

PCR

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

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

9/17

PCR

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

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

9/18

OE-PCR

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

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

9/18

Ligation

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

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

9/18

Inverse PCR

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

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

9/18

SLiCE

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

9/19

OE-PCR

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

9/19

Colony PCR

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

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

9/19

Inverse PCR

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

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

9/19

PCR

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

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

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

9/19

Colony PCR

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


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