Difference between revisions of "Team:Kyoto/Notebook"
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<p class="keln_note">Store PCR product at 4℃.</p> | <p class="keln_note">Store PCR product at 4℃.</p> | ||
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<!-- Table Generated by KELN Author: Tamukai--> | <!-- Table Generated by KELN Author: Tamukai--> | ||
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<p class="keln_note">add 4, 8 μl (add two by two to sample)<br>add 4, 8μl Enzyme master mix (add two by two to sample)<dr>digest 37℃ 30min<br>70℃ 20min<br>add 1,1.6 μl NaOAc pH5.2, 3M(add two by two to sample)<br>20, 40μl (add two by two to sample) 99.5% EtOH<dr>Vortex<br>preserve in -20℃ </p> | <p class="keln_note">add 4, 8 μl (add two by two to sample)<br>add 4, 8μl Enzyme master mix (add two by two to sample)<dr>digest 37℃ 30min<br>70℃ 20min<br>add 1,1.6 μl NaOAc pH5.2, 3M(add two by two to sample)<br>20, 40μl (add two by two to sample) 99.5% EtOH<dr>Vortex<br>preserve in -20℃ </p> | ||
| + | <!------------ Table END ------------> | ||
| + | <!-- Table Generated by KELN Author: Tsuzuki --> | ||
| + | <div class="keln_container"> | ||
| + | <a name="0712" class = "kyoto-jump"></a> | ||
| + | <span class="keln_date"><h3>7/12</h3></span> | ||
| + | <span class="keln_exp"><h4>OE-PCR</h4></span> | ||
| + | <span class="keln_researcher">Nambu</span> | ||
| + | <table class="keln_table"> | ||
| + | <tr><th>sample</th><th>KOD one PCR master mix (ul)</th><th>TDH promoter</th><th>volume (ul)</th><th>ORF</th><th>volume (ul)</th><th>CYC terminator</th><th>volume (ul)</th><th>DW (ul)</th></tr> | ||
| + | <tr><td>A</td><td>12.5</td><td>5</td><td>1.5</td><td>GFP</td><td>1.5</td><td>6</td><td>1.5</td><td>8.0</td></tr> | ||
| + | <tr><td>B</td><td>12.5</td><td>7</td><td>1.5</td><td>RFP</td><td>1.5</td><td>8</td><td>1.5</td><td>8.0</td></tr> | ||
| + | <tr><td>C</td><td>12.5</td><td>5</td><td>1.5</td><td>GFP</td><td>1.5</td><td>6</td><td>1.5</td><td>8.0</td></tr> | ||
| + | <tr><td>D</td><td>12.5</td><td>7</td><td>1.5</td><td>RFP</td><td>1.5</td><td>8</td><td>1.5</td><td>8.0</td></tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <p class="keln_note">PCR <br>98℃ 0:30<br>98℃* 0:10<br>45℃* 0:05<br>68℃* 0:05<br>68℃ 2:00<br>10℃ <br>(* x 30 cycle) </p> | ||
<!------------ Table END ------------> | <!------------ Table END ------------> | ||
Revision as of 00:39, 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)