Difference between revisions of "Team:Kyoto/Notebook"
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<span class="keln_exp"><h4>Miniprep_Nanodrop</h4></span> | <span class="keln_exp"><h4>Miniprep_Nanodrop</h4></span> | ||
<span class="keln_researcher">Nambu</span> | <span class="keln_researcher">Nambu</span> | ||
| − | |||
| − | |||
| − | |||
</div> | </div> | ||
<p class="keln_note">sample:<br>505,506,507,508,516,522,530</p> | <p class="keln_note">sample:<br>505,506,507,508,516,522,530</p> | ||
<!------------ Table END ------------> | <!------------ Table END ------------> | ||
Revision as of 10:53, 6 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 | 31 | ||
| 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
| 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)
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℃
Transformation
See "Protocol" Page
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.
sample:
505,506,507,508,516,522,530