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<!-- Table Generated by KELN Author: Tsuzuki --> | <!-- Table Generated by KELN Author: Tsuzuki --> | ||
<div class="keln_container"> | <div class="keln_container"> | ||
− | <span class="keln_date"><h3><a name="0705 | + | <span class="keln_date"><h3><a name="0705">7/5</a></h3></span> |
<span class="keln_exp"><h4>OE-PCR</h4></span> | <span class="keln_exp"><h4>OE-PCR</h4></span> | ||
<span class="keln_researcher">Nambu, Shimazoe</span> | <span class="keln_researcher">Nambu, Shimazoe</span> |
Revision as of 15:11, 10 September 2018
Notebook
July | ||||||
---|---|---|---|---|---|---|
1 | ||||||
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, Tamukaicomponent | volume(μl) |
---|---|
BSA | 0.5 |
buffer(10xH) | 5 |
EcoR1 | 0.5 |
Pst1 | 0.5 |
Dpn1 | 0.5 |
dH2O | 18 |
add 4μl linearized plasmid backbone (25ng/μl)
add 4μl Enzyme Master Mix
digest 37℃, 30min
add 8μl NaOAc (pH5.2,3M)
add 16μl 99.5% EtOh
Vortex 2sec
add 184μl 99.5% EtOH
add 72μl DW
7/5
OE-PCR
Nambu, Shimazoesample | Primer Forward | volume (ul) | Primer Reverse | volume (ul) | template DNA | volume (ul) | Q5 HF 2xMasterMix (ul) | DW (ul) |
---|---|---|---|---|---|---|---|---|
1 | Z007 | 1.25 | Z009 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
2 | Z008 | 1.25 | Z011 | 1.25 | GFP | 1.0 | 1.0 | 9.0 |
3 | Z010 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
4 | Z007 | 1.25 | Z014 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
5 | Z013 | 1.25 | Z016 | 1.25 | RFP | 1.0 | 1.0 | 9.0 |
6 | Z015 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
7/6
OE-PCR
Nambu, Shimazoesample | Primer Forward | volume (ul) | Primer Reverse | volume (ul) | Template DNA | volume (ul) | Q5 HF2x Master Mix (ul) | DW (ul) |
---|---|---|---|---|---|---|---|---|
1 | Z007 | 1.25 | Z009 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
2 | Z010 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
3 | Z007 | 1.25 | Z014 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
4 | Z015 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
5* | Z007 | 1.25 | Z009 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
6* | Z010 | 1.25 | Z012 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
7* | Z007 | 1.25 | Z014 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
8* | Z015 | 1.25 | Z012 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
*sample5~8 x 2set
7/6
colony_PCR
Mangrin colony | DW (ul) | Q5 HF2x MasterMix (ul) | m13 Forward (ul) | m13 Reverse (ul) |
---|---|---|---|---|
1 | 2.0 | 2.5 | 0.25 | 0.25 |
2 | 2.0 | 2.5 | 0.25 | 0.25 |
3 | 2.0 | 2.5 | 0.25 | 0.25 |
4 | 2.0 | 2.5 | 0.25 | 0.25 |
5 | 2.0 | 2.5 | 0.25 | 0.25 |
Store PCR product at 4℃.
7/10
Restriction Enzyme Digestion
Tong, Tamukaicomponent | volume(μl) |
---|---|
BSA | 0.5 |
buffer(10xH) | 5 |
EcoR1 | 0.5 |
Pst1 | 0.5 |
Dpn1 | 0.5 |
dH2O | 18 |
add 4, 8 μl (add two by two to sample)
add 4, 8μl Enzyme master mix (add two by two to sample)
70℃ 20min
add 1,1.6 μl NaOAc pH5.2, 3M(add two by two to sample)
20, 40μl (add two by two to sample) 99.5% EtOH
preserve in -20℃
7/12
OE-PCR
Nambusample | KOD one PCR master mix (ul) | TDH promoter | volume (ul) | ORF | volume (ul) | CYC terminator | volume (ul) | DW (ul) |
---|---|---|---|---|---|---|---|---|
A | 12.5 | 5 | 1.5 | GFP | 1.5 | 6 | 1.5 | 8.0 |
B | 12.5 | 7 | 1.5 | RFP | 1.5 | 8 | 1.5 | 8.0 |
C | 12.5 | 5 | 1.5 | GFP | 1.5 | 6 | 1.5 | 8.0 |
D | 12.5 | 7 | 1.5 | RFP | 1.5 | 8 | 1.5 | 8.0 |
PCR
98℃ 0:30
98℃* 0:10
45℃* 0:05
68℃* 0:05
68℃ 2:00
10℃
(* x 30 cycle)
Mix 10μl of Competent Cell and 2μl G,R Ligation Product on ice
On ice for 30min
Heat Shock 42℃ for 45sec
On ice for 1,2 min
Add 100μl SOC medium and culture in 37℃ for 1h
Spread on a plate (50μl X-gal added)
Add 0.6μl 10x Buffer to all sample (1~14)
Culture in 10mL
8/7
YPD
TongComponent | (1L) | (900mL) | (600mL) |
---|---|---|---|
Peptone | 20g | 18g | 12g |
Yeast Extract | 10g | 9g | 6g |
Agar | 15g | 13.5g | 9g |
DW | xmL | 810ml | 480mL |
Glucose+DW | 20g+ymL (x+y=1000) | 18g | 12g |
fluid culture
cultured 9 yeast strains in liquid YPD.
Put 15 ml of YPD medium in a 50 ml sterile tube
push the yeast with a white chip, put it in the tube
loosen the lid and put it in the shaker
For each 8 samples, transferred to a 1.5 ml tube 1mL each.
For soy sauce yeast only 15 ml was centrifuged, decantation was repeated and scaled down to 1 ml.
8/13
Ligation
NambuNumber | 1 | 2 | 3 | 4 | 5 | 6 |
---|---|---|---|---|---|---|
vector | 426 | 316 | 426 | 316 | 426 | 316 |
insert | Mangrin | Mangrin | ZrFPS1-2 | ZrFPS1-2 | McHKT2 | McHKT2 |
Mix the following material (Contamination are shown in the table above)
Plasmid(vector): 0.5µL
Insert: 1.0µl
Ligation High: 1.5µl
Put in Heatblock
Normal | null |
---|---|
Tryptone | 4g |
Yeast Extract | 2g |
Agar | 4g |
Glucose | 4g |
Make
25% NaCl YPD solid medium 400 mL
Normal YPD solid medium 200 mL
YPD liquid medium 500 mL
8/14
Transformation
TamukaiSample Name | Sample Volume (μl) | Competent Cells/(μl) |
---|---|---|
Mangrin | 3 | 15 |
ZrFPS1-2 | 3 | 15 |
McHKT2 | 3 | 15 |
PCR Colony
98℃ 0:30 number Backbone Insert
98℃* 0:10 301~306 SmaCIP AtHKT1
57℃* 0:30 307~317 SmaCIP SseNHX1
72℃* 1:00 318~326 SmaCIP ZrFPs1-1st
72℃ 2:00 327~337 SmaCIP AtNHXS1
4℃ ∞ 338~356 SmaCIP FgβF3
(*× 30 cycle)
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
mix the following material
SmaCIP (Backbone) : 1ul
gBlock (Insert) : 1ul
Ligation High : 2ul
(Total : 4ul)
putin HeatBlock overnight at 18℃
8/19
PCR
Nambusample | content (gBlock) | primer1 | primer2 |
---|---|---|---|
1 | Mangrin | Z026 | Z027 |
2 | ZrFPS1_2nd | Z026 | Z027 |
3 | McHKT2 | Z026 | Z027 |
4 | AtHKT1 | Z026 | Z027 |
5 | ZrFPS1_1st | Z026 | Z027 |
6 | AtNHXS1 | Z026 | Z027 |
7 | SseNHX1 | Z026 | Z027 |
8 | AVP1_1st + AVP1_2nd | Z026 | Z027 |
94℃ 2:00
98℃* 0:10
48℃ *0:30
68℃ *3:00
68℃ 0:10
10℃
* x 30 cycle
8/20
Ligation
Nambunumber | gBlock (Insert) | volume (ul) | SmaCIP (backbone) (ul) | Ligation High (ul) |
---|---|---|---|---|
7 | SdrG | 1 | 1 | 2 |
8 | SdrG-Fg overlap | 1 | 1 | 2 |
9 | ZrGPD1 | 1 | 1 | 2 |
Put in Incubator overnight at 18℃.
8/20
Transformation
NambuSample Name | Sample Volume (μl) | Competent Cells/(μl) | Medium |
---|---|---|---|
Mangrin | 4 | 16 | SOC Medium |
ZrFPS1_2nd | 4 | 16 | SOC Medium |
McHKT2 | 4 | 16 | SOC Medium |
AtHKT1 | 4 | 16 | SOC Medium |
ZrFPS1_1st | 4 | 16 | SOC Medium |
AtNHXS1 | 4 | 16 | SOC Medium |
8/21
Restriction Enzyme Digestion
Shimazoeplasmid | plasmid volume (ul) | DW (ul) | 10x Fast digest buffer (ul) | EcoR1 (ul) | Pst1 (ul) | Total (ul) |
---|---|---|---|---|---|---|
17 | 2 | 14 | 2 | 1 | 1 | 20 |
19 | 1 | 14 | 2 | 1 | 1 | 20 |
26 | 2 | 14 | 2 | 1 | 1 | 20 |
110 | 2 | 14 | 2 | 1 | 1 | 20 |
120 | 2 | 14 | 2 | 1 | 1 | 20 |
8/21
Digest plasmid
Tongplasmid | plasmid volume (ul) | DW (ul) | 10x Fast Digest Buffer (ul) | EcoR1 (ul) | Pst1 (ul) |
---|---|---|---|---|---|
304 | 2 | 14 | 2 | 1 | 1 |
309 | 2 | 14 | 2 | 1 | 1 |
313 | 2 | 14 | 2 | 1 | 1 |
314 | 2 | 14 | 2 | 1 | 1 |
317 | 2 | 14 | 2 | 1 | 1 |
325 | 2 | 14 | 2 | 1 | 1 |
342 | 2 | 14 | 2 | 1 | 1 |
322 | 2 | 14 | 2 | 1 | 1 |
336 | 2 | 14 | 2 | 1 | 1 |
343 | 2 | 14 | 2 | 1 | 1 |
352 | 2 | 14 | 2 | 1 | 1 |
353 | 2 | 14 | 2 | 1 | 1 |
354 | 2 | 14 | 2 | 1 | 1 |
15min 37℃ incubate
8/25
OE-PCR
Shimazoeplasmid | plasmid volume (ul) | DW (ul) | Q5 (ul) | primer1 | primer1 volume (ul) | primer2 | primer2 volume (ul) |
---|---|---|---|---|---|---|---|
38 (AVP1_1st) | 0.5 | 3.5 | 5 | Z064 | 0.5 | Z026(p) | 0.5 |
83 (AVP1_2nd) | 0.5 | 3.5 | 5 | Z065 | 0.5 | Z027(p) | 0.5 |
8/25
colony_PCR
Shimazoesample | Q5 (ul) | DW (ul) | primer m13f (ul) | primer m13r (ul) |
---|---|---|---|---|
38 | 2.5 | 2.0 | 0.25 | 0.25 |
83 | 2.5 | 2.0 | 0.25 | 0.25 |
colony PCR x 2 set
8/26
Liquid Culture
Nambusample | LB (ml) | Amp(100mg/ml) (ul) |
---|---|---|
505 | 50 | 50 |
506 | 50 | 50 |
507 | 50 | 50 |
508 | 50 | 50 |
516 | 50 | 50 |
522 | 50 | 50 |
530 | 50 | 50 |
Put them in incubator at 37℃ for 19 hours.
sample:505,506,507,508,516,522,530
PCR
98℃* 10s
50℃* 5s
68℃* 20s
68℃ 1min
4℃ ∞
(*× 40 cycle)
PCR
98℃* 10s
50℃* 5s
68℃* 20s
68℃ 1min
4℃ ∞
(*× 40 cycle)
8/28
OE-PCR
ShimazoeComponent | Amount(μl) |
---|---|
DW | 20 |
Z026℗ | 1.5 |
Z027℗ | 1.5 |
KOD one | 25 |
AVP1-1st | 1 |
AVP1-2nd | 1 |
Total | 50 |
PCR
98℃* 10s
65℃* 5s
68℃* 30s
68℃ 1min
4℃ ∞
(*× 25 cycle )
8/28
Colony PCR
ShimazoeComponent | Amount(μl) |
---|---|
2× Gflex PCR Buffer | 162.5 |
primer m13f | 6.5 |
primer m13r | 6.5 |
Tks Gflex DNA polymerase | 6.5 |
DW | 143 |
Colony | Poke |
Total | 325 |
PCR
number
94℃ 1min SdrG 601~611
98℃* 10s Sdr-Fg 612~623
55℃* 15s ZrGPD1 624~631
68℃* 2min Mangrin 633~638
4℃ ∞ Zr-FPS1-2nd 639~642
(*× 30 cycle) McHKT2 643~649
ZrFPS1-1st 650~657
8/29
PCR
ShimazoeComponent | Amount(μl) |
---|---|
5× Q5 Reaction Buffer | 15 |
10mM dNTPs | 1.5 |
10uM Z041 | 3.75 |
10uM Z042 | 3.75 |
Template | Poke |
Q5 DNA Polymerase | 0.75 |
DW | 47.25 |
Total | 72 |
Template
AtHKT1(304), SseNHXS1(317), AtNHXS1(336)
PCR
98℃ 30s
98℃* 10s
65℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)
8/29
PCR
ShimazoeComponent | Amount(μl) |
---|---|
5× Q5 Reaction Buffer | 5 |
10mM dNTPs | 0.5 |
10uM Z041 | 1.25 |
10uM Z042 | 1.25 |
Template | Poke |
Q5 DNA Polymerase | 0.25 |
DW | 16.75 |
Total | 24 |
Template
FgβF3
PCR
98℃ 30s
98℃* 10s
65℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)
8/30
Reaction Enzyme Digestion
Shimazoeplasmid | plasmid(ul) | 10× T(ul) | 10× BSA(ul) | SmaⅠ(ul) | CIAP(ul) | DW(ul) | Total/(ul) |
---|---|---|---|---|---|---|---|
426 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
316 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
425 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
315 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
423 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
313 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
6 sample
37℃ overnight
8/30
OE-PCR
Shimazoesample | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | Z026-Z039 (ul) | Z040-Z027 (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) | |
---|---|---|---|---|---|---|---|---|---|---|---|
A | AtHKT1 | 0.5 | 2 | 0.2 | 0.5 | 0.5 | 0.5 | 0.5 | 0.1 | 5.2 | 10 |
B | SseNHX1 | 0.5 | 2 | 0.2 | 0.5 | 0.5 | 0.5 | 0.5 | 0.1 | 5.2 | 10 |
C | AtNHXS1 | 0.5 | 2 | 0.2 | 0.5 | 0.5 | 0.5 | 0.5 | 0.1 | 5.2 | 10 |
PCR
98℃ 30s
98℃* 10s
72℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 25 cycle)
8/30
PCR
Shimazoetemplate | volume (ul) | primer① | volume (ul) | primer② | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
---|---|---|---|---|---|---|---|---|---|---|
AVP1-1st | 0.5 | 10uM Z026℗ | 0.5 | 10uM Z064 | 0.5 | 2 | 0.2 | 0.1 | 6.2 | 10 |
AVP1-2nd | 0.5 | 10uM Z027℗ | 0.5 | 10uM Z065 | 0.5 | 2 | 0.2 | 0.1 | 6.2 | 10 |
PCR
98℃ 30s
98℃* 10s
67℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 25 cycle)
8/30
OE-PCR
Shimazoenumber | template | volume (ul) | 10uM Z026℗ | 10uM Z027℗ | Z026-Z039 (ul) | Z040-Z027 (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
---|---|---|---|---|---|---|---|---|---|---|---|
A | AtHKT1 | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
B | SseNHX1 | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
C | AtNHXS1 | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
D | GFP | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
E | RFP | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
PCR
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
8/30
PCR
Shimazoetemplate | volume (ul) | 10uM primer① | volume (ul) | 10uM primer② | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
---|---|---|---|---|---|---|---|---|---|---|
AVP1-1st (PCR products) | 1 | Z026℗ | 1.25 | Z064 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
AVP1-1st (Plasmid) | 1 | Z026℗ | 1.25 | Z064 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
AVP1-2nd (PCR products) | 1 | Z027℗ | 1.25 | Z065 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
AVP1-2nd (Plasmid) | 1 | Z027℗ | 1.25 | Z065 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
PCR
98℃ 30s
98℃* 10s
65℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)
8/31
OE-PCR
sample | template | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | 10uM Z026℗ | 10uM Z027℗ | Z026-Z039 (ul) | Z040-Z027 (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
---|---|---|---|---|---|---|---|---|---|---|---|
A | AtHKT1 | 5 | 10 | 1 | 2.5 | 2.5 | 5 | 5 | 0.5 | 18.5 | 50 |
B | AtNHXS1 | 5 | 10 | 1 | 2.5 | 2.5 | 5 | 5 | 0.5 | 18.5 | 50 |
C | GFP | 5 | 10 | 1 | 2.5 | 2.5 | 5 | 5 | 0.5 | 18.5 | 50 |
D | RFP | 5 | 10 | 1 | 2.5 | 2.5 | 5 | 5 | 0.5 | 18.5 | 50 |
PCR
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1m30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
8/31
PCR
template | volume (ul) | 10uM primer① | volume (ul) | 10uM primer② | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) | |
---|---|---|---|---|---|---|---|---|---|---|---|
1 | AVP1-2nd (PCR products) | 1 | Z065 | 1.25 | Z027℗ | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
2 | AVP1-2nd (Plasmid) | 1 | Z065 | 1.25 | Z027℗ | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
3 | GFP (PCR2) | 1 | Z026℗ | 1.25 | Z027℗ | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
4 | RFP (PCR5) | 1 | Z026℗ | 1.25 | Z027℗ | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
PCR
98℃ 30s
98℃* 10s
55℃* 30s
72℃* 1min
72℃ 2min
4℃ ∞
(*× 30 cycle)
8/31
OE-PCR
Shimazoesample | template | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | Z026-Z039 (ul) | Z040-Z027 (ul) | KOD one (ul) | DW (ul) | Total (ul) |
---|---|---|---|---|---|---|---|---|---|
A | SseNHX1 | 1 | 0.3 | 0.3 | 1 | 1 | 5 | 1.4 | 10 |
B | GFP | 1 | 0.3 | 0.3 | 1 | 1 | 5 | 1.4 | 10 |
C | RFP | 1 | 0.3 | 0.3 | 1 | 1 | 5 | 1.4 | 10 |
PCR
98℃* 10s
50℃* 5s
68℃* 30s
68℃ 30s
(*× 30 cycle)
9/1
Ligation
ShimazoeNumber | 1 | 2 | 3 | 4 |
---|---|---|---|---|
vector | pRS 426 | pRS 316 | pRS 423 | pRS 313 |
insert | AtHKT1 | AtHKT1 | AtNHXS1 | AtNHXS1 |
Mix the following material (Contamination are shown in the table above)
Plasmid(vector): 1.0ul
Insert: 1.0ul
Ligation High: 2.0ul
Put in Heatblock
9/1
OE-PCR
Shimazoetemplate | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | Z026-Z039 (ul) | Z040-Z027 (ul) | Q5 (ul) | DW (ul) | Total (ul) |
---|---|---|---|---|---|---|---|---|
SseNHX1 | 5 | 1.5 | 1.5 | 5 | 5 | 25 | 7 | 50 |
PCR
98℃ 30s
98℃* 10s
50℃* 20s
72℃* 1min30s
72℃ 2min
4℃ ∞
(*× 30 cycle)
9/1
OE-PCR
Shimazoetemplate | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
---|---|---|---|---|---|---|---|---|
AVP1-1st | 1 | 1.25 | 1.25 | 5 | 0.5 | 0.25 | 14.75 | 25 |
AVP1-2nd | 1 | 1.25 | 1.25 | 5 | 0.5 | 0.25 | 14.75 | 25 |
PCR
98℃ 30s
98℃* 10s
55℃* 20s
72℃* 2min
72℃ 2min
(*× 30 cycle)