Difference between revisions of "Team:Kyoto/Notebook"
| Line 341: | Line 341: | ||
<span class="keln_researcher">Tong, Tamukai</span> | <span class="keln_researcher">Tong, Tamukai</span> | ||
<table class="keln_table"> | <table class="keln_table"> | ||
| − | <tr><th>component</th><th>volume( | + | <tr><th>component</th><th>volume(μl)</th></tr> |
<tr><td>BSA</td><td>0.5</td></tr> | <tr><td>BSA</td><td>0.5</td></tr> | ||
<tr><td>buffer(10xH)</td><td>5</td></tr> | <tr><td>buffer(10xH)</td><td>5</td></tr> | ||
| Line 350: | Line 350: | ||
</table> | </table> | ||
</div> | </div> | ||
| − | <p class="keln_note">add | + | <p class="keln_note">add 4μl linearized plasmid backbone (25ng/μl)<br>add 4μl Enzyme Master Mix<br>digest 37℃, 30min<br>add 8μl NaOAc (pH5.2,3M)<br>add 16μl 99.5% EtOh<br>Vortex 2sec<br>add 184μl 99.5% EtOH<br>add 72μl DW</p> |
<!------------ Table END ------------> | <!------------ Table END ------------> | ||
<!-- Table Generated by KELN Author: Tsuzuki --> | <!-- Table Generated by KELN Author: Tsuzuki --> | ||
Revision as of 00:26, 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℃.
7/12
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)