Difference between revisions of "Team:Kyoto/Notebook"
| Line 627: | Line 627: | ||
</div> | </div> | ||
<p class="keln_note">94℃ 2:00<br>98℃* 0:10<br>48℃ *0:30<br>68℃ *3:00<br>68℃ 0:10<br>10℃<br>* x 30 cycle</p> | <p class="keln_note">94℃ 2:00<br>98℃* 0:10<br>48℃ *0:30<br>68℃ *3:00<br>68℃ 0:10<br>10℃<br>* x 30 cycle</p> | ||
| + | <!------------ Table END ------------> | ||
| + | <!-- Table Generated by KELN Author: Tsuzuki --> | ||
| + | <div class="keln_container"> | ||
| + | <a name="0820" class = "kyoto-jump"></a> | ||
| + | <span class="keln_date"><h3>8/20</h3></span> | ||
| + | <span class="keln_exp"><h4>Ligation</h4></span> | ||
| + | <span class="keln_researcher">Nambu</span> | ||
| + | <table class="keln_table"> | ||
| + | <tr><th>number</th><th>gBlock (Insert)</th><th>volume (ul)</th><th>SmaCIP (backbone) (ul)</th><th>Ligation High (ul)</th></tr> | ||
| + | <tr><td>7</td><td>SdrG</td><td>1</td><td>1</td><td>2</td></tr> | ||
| + | <tr><td>8</td><td>SdrG-Fg overlap</td><td>1</td><td>1</td><td>2</td></tr> | ||
| + | <tr><td>9</td><td>ZrGPD1</td><td>1</td><td>1</td><td>2</td></tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <p class="keln_note">Put in Incubator overnight at 18℃.</p> | ||
<!------------ Table END ------------> | <!------------ Table END ------------> | ||
Revision as of 09:39, 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℃.