Template:Kyoto/Notebook/DMS/23

From 2014.igem.org

(Difference between revisions)
(Created page with "<html> <div class="kyoto-notebook"> <!-- 警告：ここより上を消したら怒る --> <!--ここから春実験--> <div> <a name="0210" class="kyoto-jump"></a> <h3>2...")
Line 121: Line 121:
<tr>
<tr>
<td>1</td>
<td>1</td>
-
<td>1kbp Kabber(2&micro;L)</td>
+
<td>1kbp ladder(2&micro;L)</td>
</tr>
</tr>
<tr>
<tr>
Line 191: Line 191:
</tr>
</tr>
<tr>
<tr>
-
<td>2/12 J23118(A) Pcon</td>
+
<td>2/12 J23119(A) Pcon</td>
<td>1</td>
<td>1</td>
<td>Self-Making Competent Cells (DH5&#945;)</td>
<td>Self-Making Competent Cells (DH5&#945;)</td>
Line 228: Line 228:
<tr>
<tr>
<th>Quick Taq/(&micro;L)</th>
<th>Quick Taq/(&micro;L)</th>
-
<th>VF2/(&micro;L)</th>
+
<th>VF/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
Line 255: Line 255:
</tr>
</tr>
<tr>
<tr>
-
<td>2min</td>
+
<td>5min</td>
<td>30sec</td>
<td>30sec</td>
<td>30sec</td>
<td>30sec</td>
Line 285: Line 285:
<tr>
<tr>
<td>5</td>
<td>5</td>
-
<td>2/12 I712074(Ak) T7</td>
+
<td>T7</td>
</tr>
</tr>
</table>
</table>
Line 387: Line 387:
</tr>
</tr>
<tr>
<tr>
-
<td>9/8 J23100 2100bp</td>
+
<td>9/8 J23100 325&micro;g/ml</td>
<td>6</td>
<td>6</td>
<td>-</td>
<td>-</td>
Line 400: Line 400:
</tr>
</tr>
<tr>
<tr>
-
<td>9/8 J23100 2100bp</td>
+
<td>9/8 J23100 325&micro;g/ml</td>
<td>0.3</td>
<td>0.3</td>
<td>-</td>
<td>-</td>
Line 413: Line 413:
</tr>
</tr>
<tr>
<tr>
-
<td>9/28 RBS-GFP-DT</td>
+
<td>9/28 RBS-GFP-DT 215.9&micro;g/ml</td>
<td>9.3</td>
<td>9.3</td>
<td>-</td>
<td>-</td>
Line 453: Line 453:
<tr>
<tr>
<td>2</td>
<td>2</td>
-
<td>9/8 G23100 S,P</td>
+
<td>9/8 J23100 S,P</td>
</tr>
</tr>
<tr>
<tr>
<td>3</td>
<td>3</td>
-
<td>9/8 G23100 control</td>
+
<td>9/8 J23100 control</td>
</tr>
</tr>
<tr>
<tr>
Line 478: Line 478:
<tr>
<tr>
<td>1</td>
<td>1</td>
-
<td>9/2 J23100 S,P</td>
+
<td>9/8 J23100 S,P</td>
</tr>
</tr>
<tr>
<tr>
Line 516: Line 516:
<th>Total/(&micro;L)</tr>
<th>Total/(&micro;L)</tr>
<tr>
<tr>
-
<td>3/4 J23100 S,P 2100bp</td>
+
<td>3/4 J23100 S,P</td>
<td>4.8</td>
<td>4.8</td>
-
<td>3/4 RBS-GFP-DT X,P 900bp</td>
+
<td>3/4 RBS-GFP-DT X,P</td>
<td>9.2</td>
<td>9.2</td>
<td>Reaction Buffer</td>
<td>Reaction Buffer</td>
Line 575: Line 575:
<table>
<table>
<tr>
<tr>
-
<th>Quick Tag/(&micro;L)</th>
+
<th>Quick Taq/(&micro;L)</th>
-
<th>VF2/(&micro;L)</th>
+
<th>VF/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
Line 586: Line 586:
<td>2.6</td>
<td>2.6</td>
<td>59.8</td>
<td>59.8</td>
-
<td130</td>
+
<td>130</td>
</tr>
</tr>
</table>
</table>
Line 606: Line 606:
<td>30sec</td>
<td>30sec</td>
<td>30sec</td>
<td>30sec</td>
-
<td>24sec</td>
+
<td>72sec</td>
</tr>
</tr>
</table>
</table>
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<tr>
<tr>
<td>5</td>
<td>5</td>
-
<td>3/5 J712074 (1)</td>
+
<td>3/5 I712074 (1)</td>
</tr>
</tr>
<tr>
<tr>
<td>6</td>
<td>6</td>
-
<td>3/5 J712074 (2)</td>
+
<td>3/5 I712074 (2)</td>
</tr>
</tr>
<tr>
<tr>
<td>7</td>
<td>7</td>
-
<td>3/5 J712074 (3)</td>
+
<td>3/5 I712074 (3)</td>
</tr>
</tr>
<tr>
<tr>
<td>8</td>
<td>8</td>
-
<td>3/5 J712074 (4)</td>
+
<td>3/5 I712074 (4)</td>
</tr>
</tr>
</table>
</table>
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<tr>
<tr>
<th>Quick Taq/(&micro;L)</th>
<th>Quick Taq/(&micro;L)</th>
-
<th>VF2/(&micro;L)</th>
+
<th>VF/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
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</tr>
</tr>
<tr>
<tr>
-
<td>94&deg;C</td>
+
<td>97&deg;C</td>
<td>94&deg;C</td>
<td>94&deg;C</td>
<td>55&deg;C</td>
<td>55&deg;C</td>
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</tr>
</tr>
<tr>
<tr>
-
<td>2min</td>
+
<td>5min</td>
<td>30sec</td>
<td>30sec</td>
<td>30sec</td>
<td>30sec</td>
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<a name="0312" class="kyoto-jump"></a>
<a name="0312" class="kyoto-jump"></a>
<h3>3/12</h3>
<h3>3/12</h3>
-
+
 
-
<h4>Electrophoresis</h4>
+
<h4>Electrophoresis</h4>
<span class="kyoto-author">Shimazaki</span>
<span class="kyoto-author">Shimazaki</span>
<table>
<table>
Line 939: Line 939:
</tr>
</tr>
</table>
</table>
-
+
<h4>Colony PCR</h4>
-
<h4>Colony PCR</h4>
+
<span class="kyoto-author">Shimazaki</span>
<span class="kyoto-author">Shimazaki</span>
<p>Master Mix</p>
<p>Master Mix</p>
Line 946: Line 945:
<tr>
<tr>
<th>Quick Taq/(&micro;L)</th>
<th>Quick Taq/(&micro;L)</th>
-
<th>VF2/(&micro;L)</th>
+
<th>VF/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
Line 1,026: Line 1,025:
</tr>
</tr>
</table>
</table>
 +
</div>
</div>
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<tr>
<tr>
<td>4</td>
<td>4</td>
-
<td>3/10 pSB1C3 (1)</td>
+
<td>3/10 pSB1C3 (3)</td>
</tr>
</tr>
<tr>
<tr>
<td>5</td>
<td>5</td>
-
<td>3/10 pSB1C3 (2)</td>
+
<td>3/10 pSB1C3 (4)</td>
</tr>
</tr>
<tr>
<tr>
<td>6</td>
<td>6</td>
-
<td>3/10 DT (1)</td>
+
<td>3/10 DT (5)</td>
</tr>
</tr>
<tr>
<tr>
<td>7</td>
<td>7</td>
-
<td>3/10 DT (2)</td>
+
<td>3/10 DT (6)</td>
</tr>
</tr>
<tr>
<tr>
<td>8</td>
<td>8</td>
-
<td>3/10 T7 (1)</td>
+
<td>3/10 T7 (7)</td>
</tr>
</tr>
<tr>
<tr>
<td>9</td>
<td>9</td>
-
<td>3/10 T7 (2)</td>
+
<td>3/10 T7 (8)</td>
</tr>
</tr>
<tr>
<tr>
Line 1,096: Line 1,096:
<table>
<table>
<tr>
<tr>
-
<th>Quick Tag/(&micro;L)</th>
+
<th>Quick Taq/(&micro;L)</th>
-
<th>VF2/(&micro;L)</th>
+
<th>VF/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
Line 1,180: Line 1,180:
<table>
<table>
<tr>
<tr>
-
<th>Quick Tag/(&micro;L)</th>
+
<th>Quick Taq/(&micro;L)</th>
-
<th>VF2/(&micro;L)</th>
+
<th>VF/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
Line 1,430: Line 1,430:
<h3>3/19</h3>
<h3>3/19</h3>
 +
　　　　　<h4>Electrophoresis</h4>
 +
<span class="kyoto-author">Yoshida</span>
 +
<table>
 +
<tr>
 +
<th>Lane</th>
 +
<th>Sample</th>
 +
</tr>
 +
<tr>
 +
<td>1</td>
 +
<td>1kbp ladder</td>
 +
</tr>
 +
                                <tr>
 +
<td>2</td>
 +
<td>pSB1C3 E,X</td>
 +
</tr>
 +
                                <tr>
 +
<td>3</td>
 +
<td>pSB1C3 control</td>
 +
</tr>
 +
</table>
 +
<h4>Restriction Enzyme Digestion</h4>
<h4>Restriction Enzyme Digestion</h4>
<span class="kyoto-author">Yoshida</span>
<span class="kyoto-author">Yoshida</span>
Line 1,495: Line 1,516:
<tr>
<tr>
<th>3/19 DNA Purification product/(&micro;L)</th>
<th>3/19 DNA Purification product/(&micro;L)</th>
-
<th>F/(&micro;L)</th>
+
<th>Fw/(&micro;L)</th>
-
<th>R/(&micro;L)</th>
+
<th>Rv/(&micro;L)</th>
<th>HiFi Mix/(&micro;L)</th>
<th>HiFi Mix/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
Line 1,513: Line 1,534:
<tr>
<tr>
<th>R.pomeroyi Genome/(&micro;L)</th>
<th>R.pomeroyi Genome/(&micro;L)</th>
-
<th>F/(&micro;L)</th>
+
<th>Fw/(&micro;L)</th>
-
<th>R/(&micro;L)</th>
+
<th>Rv/(&micro;L)</th>
<th>HiFi Mix/(&micro;L)</th>
<th>HiFi Mix/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
Line 1,595: Line 1,616:
<th>Lane</th>
<th>Lane</th>
<th>Sample</th>
<th>Sample</th>
 +
</tr>
 +
                                <tr>
 +
<td>1</td>
 +
<td>Blank</td>
</tr>
</tr>
<tr>
<tr>
Line 1,703: Line 1,728:
<th>F dddD 62/(&micro;L)</th>
<th>F dddD 62/(&micro;L)</th>
<th>R dddD 68/(&micro;L)</th>
<th>R dddD 68/(&micro;L)</th>
-
<th>Templete/(&micro;L)</th>
+
<th>Templete 3/24 dddD/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
<th>Total/(&micro;L)</th>
<th>Total/(&micro;L)</th>
Line 1,723: Line 1,748:
<th>Extension</th>
<th>Extension</th>
<th>Final Extension</th>
<th>Final Extension</th>
-
<th>Cooling</th>
 
</tr>
</tr>
<tr>
<tr>
Line 1,760: Line 1,784:
<tr>
<tr>
<td>dddD</td>
<td>dddD</td>
-
<td>63.7</td>
+
<td>62.7</td>
<td>1.77</td>
<td>1.77</td>
<td>1.88</td>
<td>1.88</td>
Line 1,859: Line 1,883:
</tr>
</tr>
<tr>
<tr>
-
<td>pSB1C3 3/24 (4)</td>
+
<td>3/24 PCR product dddD E,S</td>
<td>3.2</td>
<td>3.2</td>
<td>0.5</td>
<td>0.5</td>
Line 1,949: Line 1,973:
<th>Total/(&micro;L)</tr>
<th>Total/(&micro;L)</tr>
<tr>
<tr>
-
<td>pSB1C3</td>
+
<td>pSB1C3 2047bp</td>
<td>30</td>
<td>30</td>
-
<td>dddD</td>
+
<td>dddD 2545bp</td>
<td>30</td>
<td>30</td>
<td>2x Rapid Ligation Buffer</td>
<td>2x Rapid Ligation Buffer</td>
Line 1,993: Line 2,017:
</tr>
</tr>
<tr>
<tr>
-
<td>pSB1C3(4)3/25</td>
+
<td>pSB1C3(4) 3/25</td>
<td>20.1</td>
<td>20.1</td>
<td>0.5</td>
<td>0.5</td>
Line 2,155: Line 2,179:
<tr>
<tr>
<td>1</td>
<td>1</td>
-
<td>Sequence primer (1)</td>
+
<td>Fw:Sequence primer (1) 57.9/Rv:Spe1 dddD 2276bp</td>
</tr>
</tr>
<tr>
<tr>
<td>2</td>
<td>2</td>
-
<td>Sequence primer (2)</td>
+
<td>Fw:Sequence primer (2) 57.3/Rv:Spe1 dddD 1907bp</td>
</tr>
</tr>
<tr>
<tr>
<td>3</td>
<td>3</td>
-
<td>Sequence primer (3)</td>
+
<td>Fw:Sequence primer (3) 57.6/Rv:Spe1 dddD 1527bp</td>
</tr>
</tr>
<tr>
<tr>
<td>4</td>
<td>4</td>
-
<td>Sequence primer (4)</td>
+
<td>Fw:Sequence primer (4) 56.3/Rv:Spe1 dddD 1189bp</td>
</tr>
</tr>
<tr>
<tr>
<td>5</td>
<td>5</td>
-
<td>Sequence primer (5)</td>
+
<td>Fw:Sequence primer (5) 57.2/Rv:Spe1 dddD 799bp</td>
</tr>
</tr>
<tr>
<tr>
<td>6</td>
<td>6</td>
-
<td>Sequence primer (6)</td>
+
<td>Fw:Sequence primer (6) 56.4/Rv:Spe1 dddD 398bp</td>
</tr>
</tr>
<tr>
<tr>
<td>7</td>
<td>7</td>
-
<td>Sequence primer VF2</td>
+
<td>Fw:Sequence primer VF2 56.4/Rv:Spe1 dddD 266bp</td>
</tr>
</tr>
<tr>
<tr>
<td>8</td>
<td>8</td>
-
<td>F pre-dddD PCR product 62</td>
+
<td>Fw:pre-dddD PCR product 62/Rv:Spe1 dddD</td>
</tr>
</tr>
<tr>
<tr>
<td>9</td>
<td>9</td>
-
<td>Blank</td>
+
<td>Fw:Blank/Rv:Spe1 dddD</td>
</tr>
</tr>
</table>
</table>
Line 2,275: Line 2,299:
<tr>
<tr>
<th>Quick Taq/(&micro;L)</th>
<th>Quick Taq/(&micro;L)</th>
-
<th>VF2/(&micro;L)</th>
+
<th>VF/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>VR/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
<th>MilliQ/(&micro;L)</th>
Line 2,361: Line 2,385:
<a name="0331" class="kyoto-jump"></a>
<a name="0331" class="kyoto-jump"></a>
<h3>3/31</h3>
<h3>3/31</h3>
-
+
 +
<h4>Electrophoresis</h4>
 +
<span class="kyoto-author">Yasuda</span>
<table>
<table>
<tr>
<tr>

Revision as of 14:41, 4 October 2014

2/10

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
J23100 5.7 - - 0.5 0.5 10xBuffer(K) 3 - 20.3 30
RBS-GFP-DT 7.5 - 0.5 - 0.5 10xBuffer(M) 3 3 15.5 30

Electrophoresis/Gel Extraction

Yasuda
Lane Sample
1 1kbp ladder(2µL)
2 J23100 DNA control
3 J23100 S,P experimental
4 J23100 S,P experimental

Restriction Enzyme Digestion

Yasuda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
J23100 10 - - 0.5 0.5 10xBuffer(K) 3 - 16 30

2/12

Electrophoresis/Gel Extraction

Yasuda
Lane Sample
1 1kbp ladder(2µL)
2 J23100 DNA control
3 J23100 S,P experimental
4 J23100 S,P experimental
Sample Concentration/(µg/mL) 260/280 260/230
GFP 16.7 1.46 0.64
Pcon 17.2 1.69 0.26

Ligation

Inoue
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
2/10 J23100 S,P 2100bp 5.8 2/10 RBS-GFP-DT 898bp 4.9 2x Buffer 5 T4 Ligase 3 - 10

Transformation

Yasuda
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
2/12 J23119(A) Pcon 1 Self-Making Competent Cells (DH5α) 10 11 SOC
2/12 I712074 (Ak) T7 1 Self-Making Competent Cells (DH5α) 10 11 SOC
2/12 Ligation Pcon+RBS-GFP-PT 1 Self-Making Competent Cells (DH5α) 10 11 SOC

2/13

Colony PCR

Yasuda,Murakami
Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
30 1.2 1.2 27.6 60
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
5min 30sec 30sec 72sec

Denature-Annealing-Extension 30cycle

Number Sample
1 Pcon-RBS-GFP-DT (1)
2 Pcon-RBS-GFP-DT (2)
3 Pcon-RBS-GFP-DT (3)
4 Pcon-RBS-GFP-DT (4)
5 T7

Electrophoresis

Honda
Lane Sample
1 1kbp ladder
2 2/12 Pcon-RBS-GFP-DT (1)
3 2/12 Pcon-RBS-GFP-DT (2)
4 2/12 Pcon-RBS-GFP-DT (3)
5 2/12 Pcon-RBS-GFP-DT (4)
6 2/12 I712074(Ak) T7

Liquid Culture

No Name
Sample Medium
Pcon-RBS-GFP-DT Amp

2/14

Miniprep

Murakami
Sample
2/13 Pcon-RBS-GFP-DT(1)
2/13 Pcon-RBS-GFP-DT(2)

2/23

Self-Making Competent Cells (DH5α)

Tatsui and Kojima

3/4

Restriction Enzyme Digestion

Kato, Murata and Shimazaki
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
9/8 J23100 325µg/ml 6 - - 0.5 0.5 H 3 3 17 30
9/8 J23100 325µg/ml 0.3 - - - - H 1 1 7.7 10
9/28 RBS-GFP-DT 215.9µg/ml 9.3 - 0.5 - 0.5 H 3 3 13.7 30
control 0.5 - - - - H 1 1 7.5 10

Electrophoresis

Kato, Murata and Shimazaki
Lane Sample
1 1kbp ladder
2 9/8 J23100 S,P
3 9/8 J23100 control
4 9/28 RBS-GFP DT X,P
5 9/28 RBS-GFP DT control

Gel Extraction

Kato, Murata and Shimazaki
Lane Sample
1 9/8 J23100 S,P
2 Blank
3 Blank
4 9/28 RBS-GFP-DT X,P
5 1kbp ladder

3/5

Ligation

Kato, Shimazaki and Murata
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
3/4 J23100 S,P 4.8 3/4 RBS-GFP-DT X,P 9.2 Reaction Buffer 2 Ligase(Bio Lub) 1 3 20

Transformation

No Name
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
3/5 J23119 1 Self-Making Competent Cells (DH5α) 10 11 SOC
3/5 I712074 1 Self-Making Competent Cells (DH5α) 10 11 SOC
3/5 ligation Pcon RBS-GFP-DT 1 Self-Making Competent Cells (DH5α) 10 11 SOC

3/6

Colony PCR

Kato and Shimazaki
Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
65 2.6 2.6 59.8 130
Predenature Denature Annealing Extension
94°C 94°C 55°C 68°C
2min 30sec 30sec 72sec

Denature-Annealing-Extension 30cycle

Number Sample
1 3/5 J23119 (1)
2 3/5 J23119 (2)
3 3/5 J23119 (3)
4 3/5 J23119 (4)
5 3/5 I712074 (1)
6 3/5 I712074 (2)
7 3/5 I712074 (3)
8 3/5 I712074 (4)
Number Sample
1 3/5 Pcon RBS-GFP-DT (1)
2 3/5 Pcon RBS-GFP-DT (2)
3 3/5 Pcon RBS-GFP-DT (3)
4 3/5 Pcon RBS-GFP-DT (4)

Electrophoresis

Murata
Lane Sample
1 1kbp ladder
2 3/5 J23119 (1)
3 3/5 J23119 (2)
4 3/5 J23119 (3)
5 3/5 J23119 (4)
6 3/5 I712074 (1)
7 3/5 I712074 (2)
8 3/5 I712074 (3)
9 3/5 I712074 (4)
10 3/5 Pcon RBS-GFP-DT (1)
11 3/5 Pcon RBS-GFP-DT (2)
12 3/5 Pcon RBS-GFP-DT (3)
13 3/5 Pcon RBS-GFP-DT (4)
14 1kbp ladder

3/7

Miniprep

Murata
Sample
3/6 Pcon RBS-GFP-DT (1)
3/6 Pcon RBS-GFP-DT (2)

3/10

Transformation

Okazaki
Sample/(µL) CompetentCells/(µL) Medium
3/10 RBS-GFP Self-Making Competent Cells (DH5α) CP
RBS Self-Making Competent Cells (DH5α) Amp
DT Self-Making Competent Cells (DH5α) CP
3/10 T7 Self-Making Competent Cells (DH5α) Amp

3/11

Colony PCR

Shimazaki

Master Mix

Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
50 2 2 46 100
Predenature Denature Annealing Extension
97°C 94°C 55°C 68°C
5min 30sec 30sec 60sec

Denature-Annealing-Extension 30cycle

Number Sample
1 3/10 RBS (1)
2 3/10 RBS (2)
3 3/10 pSB1C3 (1)
4 3/10 pSB1C3 (2)
5 3/10 DT (1)
6 3/10 DT (2)
7 3/10 T7 (1)
8 3/10 T7 (2)
9 control

3/12

Electrophoresis

Shimazaki
Lane Sample
1 3/10 RBS (1)
2 3/10 RBS (2)
3 3/10 pSB1C3 (1)
4 3/10 pSB1C3 (2)
5 3/10 DT (1)
6 3/10 DT (2)
7 3/10 T7 (1)
8 3/10 T7 (2)
9 control
10 1kbp ladder

Colony PCR

Shimazaki

Master Mix

Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
50 2.0 2.0 46 100
Predenature Denature Annealing Extension
97°C 94°C 55°C 68°C
5min 30sec 30sec 60sec

Denature-Annealing-Extension 30cycle

Number Sample
1 3/10 RBS 3
2 3/10 RBS 4
3 3/10 pSB1C3 4
4 3/10 pSB1C3 5
5 3/10 DT 4
6 3/10 DT 5
7 3/10 T7 3
8 3/10 T7 4
9 new QuickTaq control
10 old QuickTaq control

3/14

Electrophoresis

Kojima
Lane Sample
1 1kbp ladder
2 3/10 RBS (1)
3 3/10 RBS (2)
4 3/10 pSB1C3 (3)
5 3/10 pSB1C3 (4)
6 3/10 DT (5)
7 3/10 DT (6)
8 3/10 T7 (7)
9 3/10 T7 (8)
10 old Quick Taq control (9)
11 new Quick Taq control (10)
12 1kbp ladder

Colony PCR

Kojima

Master Mix

Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
17.5 0.7 0.7 16.1(MilliQ) 35
Predenature Denature Annealing Extension
97°C 95°C 58°C 68°C
5min 30sec 30sec 30sec

Denature-Annealing-Extension 30cycle

Number Sample
1 RBS-4
2 DT-5
3 control

Electrophoresis

Kojima
Lane Sample
1 1kbp ladder
2 RBS-4
3 DT-5
4 control

Colony PCR

Yasuda and Shimazaki

Master Mix

Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
17.5 0.7 0.7 16.1(MilliQ) 35
Predenature Denature Annealing Extension
97°C 95°C 58°C 68°C
5min 30sec 30sec 60sec

Denature-Annealing-Extension 30cycle

Number Sample
1 pSB1C3-3
2 T7-5
3 control

Electrophoresis

Shimazaki
Lane Sample
1 1kbp ladder
2 pSB1C3-3
3 T7-5
4 control
4 2/10 MilliQ

3/17

Liquid Culture

Yoshida
Sample Medium
3/10 pSB1C3-3 CP
3/10 DT-5 CP
3/10 T7-5 Amp
3/10 RBS-4 Amp

3/18

Miniprep

Murata and Yasuda
Sample Concentration/(µg/mL) 260/280 260/230
3/17 DT CP 64.4 1.17 0.29
3/17 RBS Amp 61.4 1.28 0.36
3/17 T7 Amp 66.8 1.22 0.34
3/17 pSB1C3 64.2 1.31 0.39

Restriction Enzyme Digestion

Yoshida
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 E,S 3/18 miniprep product 20 1 1 - - M 3 3 2 30
control 3 - - - - M 1 1 5 10

Cloning PCR

Murata and Yasuda
2K KAPA HiFi HotStart ReadyMix/(µL) F dddD 66/(µL) R dddD 65/(µL) R.pomeroyi Genome/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 3 8 25
Predenature Denature Annealing Extension Final Extension
95°C 98°C 65°C 72°C 72°C
3min 20sec 15sec 150sec 1sec

Denature-Annealing-Extension 25cycle

3/19

　　　　　

Electrophoresis

Yoshida
Lane Sample
1 1kbp ladder
2 pSB1C3 E,X
3 pSB1C3 control

Restriction Enzyme Digestion

Yoshida
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 3/18 miniprep product 20 1 - 1 - H 3 - 5 30
control 3 - - - - H 1 - 6 10

Electrophoresis

Kojima
Lane Sample
1 1kbp ladder
2 dddD PCR product

PCR

Kojima
3/19 DNA Purification product/(µL) Fw/(µL) Rv/(µL) HiFi Mix/(µL) MilliQ/(µL) Total/(µL)
1 0.75 0.75 12.5 10 25
R.pomeroyi Genome/(µL) Fw/(µL) Rv/(µL) HiFi Mix/(µL) MilliQ/(µL) Total/(µL)
3 0.75 0.75 12.5 8 25
Predenature Denature Annealing Extension Final Extension
95°C 98°C 68°C 72°C 72°C
5min 20sec 15sec 90sec 5min

Denature-Annealing-Extension 25cycle

3/20

Electrophoresis

Kojima
Lane Sample
1 1kbp ladder
2 pSB1C3 E,S
3 control
4 dddD(1) 3/19 PCR product
5 dddD(2) 3/19 PCR product

Gel Extraction

Yasuda and Tatsui
Lane Sample
1 Blank
2 1kbp ladder
3 pSB1C3
4 Blank
5 pSB1C3

Gel Extraction

Murara
Lane Sample
1 1kbp ladder
2 dddD
3 dddD
Sample Concentration/(µg/mL) 260/280 260/230
dddD 35 1.63 0.85

3/24

Restriction Enzyme Digestion

Honda
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 11 0.5 0.5 - - M 1.5 1.5 - 15
dddD 12.5 0.5 - 0.5 - M 1.5 - - 15

PCR

Honda
2x KAPA HiFi HotStart ReadyMix/(µL) F dddD 62/(µL) R dddD 68/(µL) Templete 3/24 dddD/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 pick up 11 25
Predenature Denature Annealing Extension Final Extension
95°C 98°C 68°C 72°C 72°C
5min 20sec 15sec 90sec 5min

Denature-Annealing-Extension 25cycle

Number Sample
1 dddD
Sample Concentration/(µg/mL) 260/280 260/230
dddD 62.7 1.77 1.88

Ligation

Honda
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
dddD 1.3 pSB1C3 0.43 2xBuffer 5 T4Ligase 1 2.27 10

Liquid Culture

Tatsui
Sample Medium
pSB1C3(3) CP
pSB1C3(4) CP

3/25

Miniprep

Tatsui
Sample Concentration/(µg/mL)
pSB1C3 3/24 (3) 41
pSB1C3 3/24 (4) 57

Restriction Enzyme Digestion

Murata
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 3/24 (3) 15.4 0.5 - 0.5 - H 3 - 10.6 30
3/24 PCR product dddD E,S 3.2 0.5 - 0.5 - H 3 - 22.8 30

Gel Extraction

Murata
Lane Sample
1 1kbp ladder
2 pSB1C3(3) E,S 3/25
3 Blank
4 Blank
5 Blank
6 dddD 3/25 E,S
7 dddD 3/25 E,S
Sample Concentration/(µg/mL) 260/280 260/230
dddD 4.9 17.5 0.52
pSB1C3 2.0 - 0.21

3/26

Ligation

Yasuda
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 2047bp 30 dddD 2545bp 30 2x Rapid Ligation Buffer 5 T4Ligase 3 - 10

Transformation

Yasuda
Sample/(µL)
dddD(pSB1C3)

3/27

Restriction Enzyme Digestion

Murata
Sample/(µL) EcoR1/(µL) Xba1/(µL) Spe1/(µL) Pst1/(µL) Buffer/(µL) BSA/(µL) MilliQ/(µL) Total/(µL)
pSB1C3(4) 3/25 20.1 0.5 - 0.5 - 10xBuffer 3 - 5.9 30
dddD 3/24 PCR product 3.2 0.5 - 0.5 - 10xBuffer 3 - 22.8 30

Gel Extraction

Honda
Lane Sample
1 1kbp ladder
2 pSB1C3 E,S 3/27
3 pSB1C3 E,S 3/27
4 Blank
5 dddD E,S 3/27
6 dddD E,S 3/27
Sample Concentration/(µg/mL) 260/280 260/230
pSB1C3 9 1.58 0.04
dddD 16.0 1.78 0.08

Ligation

Honda
Vector/(µL) Insert/(µL) Buffer/(µL) Ligase/(µL) MilliQ/(µL) Total/(µL)
pSB1C3 11 dddD 15 2x Rapid Ligation Buffer 5 T4 DNA Ligase 1 4 10

3/28

PCR

Yasuda
2x KAPA HiFi HotStart ReadyMix/(µL) F Sequence primer/(µL) R Spe1-dddD/(µL) Templete/(µL) MilliQ/(µL) Total/(µL)
12.5 0.75 0.75 pick up 11 25
Predenature Denature Annealing Extension Final Extension Cooling
95°C 98°C 68°C 72°C 72°C
5min 20sec 15sec 90sec 5min

Denature-Annealing-Extension 25cycle

Number Sample
1 Fw:Sequence primer (1) 57.9/Rv:Spe1 dddD 2276bp
2 Fw:Sequence primer (2) 57.3/Rv:Spe1 dddD 1907bp
3 Fw:Sequence primer (3) 57.6/Rv:Spe1 dddD 1527bp
4 Fw:Sequence primer (4) 56.3/Rv:Spe1 dddD 1189bp
5 Fw:Sequence primer (5) 57.2/Rv:Spe1 dddD 799bp
6 Fw:Sequence primer (6) 56.4/Rv:Spe1 dddD 398bp
7 Fw:Sequence primer VF2 56.4/Rv:Spe1 dddD 266bp
8 Fw:pre-dddD PCR product 62/Rv:Spe1 dddD
9 Fw:Blank/Rv:Spe1 dddD

Electrophoresis

Murata
Lane Sample
1 1kbp ladder
2 Seaquence primer (1) PCR product
3 Seaquence primer (2) PCR product
4 Seaquence primer (3) PCR product
5 Seaquence primer (4) PCR product
6 Seaquence primer (5) PCR product
7 Seaquence primer (6) PCR product
8 Seaquence primer VF2
9 F pre-dddD cloning
10 Blank
11 1kbp ladder

Transformation

Kojima
Sample/(µL) CompetentCells/(µL) Total/(µL) Medium
dddD 1 Self-Making Competent Cells (DH5α) 10 11 CP

3/30

Colony PCR

Yasuda
Quick Taq/(µL) VF/(µL) VR/(µL) MilliQ/(µL) Total/(µL)
5 0.2 0.2 4.6 10
Predenature Denature Annealing Extension
97°C 95°C 58°C 68°C
2min 30sec 30sec 168sec

Denature-Annealing-Extension 30cycle

Number Sample
1 3/26 dddD(pSB1C3) (1)
2 3/26 dddD(pSB1C3) (2)
3 3/26 dddD(pSB1C3) (3)
4 3/26 dddD(pSB1C3) (4)
5 3/26 dddD(pSB1C3) (5)
6 3/28 dddD(pSB1C3) (6)
7 3/28 dddD(pSB1C3) (7)
8 3/28 dddD(pSB1C3) (8)
9 3/28 dddD(pSB1C3) (9)
10 3/28 dddD(pSB1C3) (10)

3/31

Electrophoresis

Yasuda
Number Sample
1 1kbp ladder
2 3/26 dddD(pSB1C3) (1)
3 3/26 dddD(pSB1C3) (2)
4 3/26 dddD(pSB1C3) (3)
5 3/26 dddD(pSB1C3) (4)
6 3/26 dddD(pSB1C3) (5)
7 1kbp ladder
8 3/28 dddD(pSB1C3) (6)
9 3/28 dddD(pSB1C3) (7)
10 3/28 dddD(pSB1C3) (8)
11 3/28 dddD(pSB1C3) (9)
12 3/28 dddD(pSB1C3) (10)
13 1kbp ladder

Retrieved from "http://2014.igem.org/Template:Kyoto/Notebook/DMS/23"