Team:KIT-Kyoto/Notebook/Labnote

From 2014.igem.org

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<div class="scroll"></div>
<div class="scroll"></div>
<div class="ac">
<div class="ac">
 +
<h3>August 19, 2014</h3>
<p class="sentence">
<p class="sentence">
 +
Conducted mutagenesis to remove an illegal restriction site (EcoR1)
<br>
<br>
 +
<strong>InversePCR</strong>
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th colspan="4">Sample</th>
 +
</tr>
 +
<tr>
 +
<th>Vector</th>
 +
<th>Insert</th>
 +
<th>Primer</th>
 +
<th>DNA polymer-ase</th>
 +
</tr>
 +
<tr>
 +
<td>pGEX6P-2</td>
 +
<td>CuMTS3</td>
 +
<td>Ter-pinene3-mutagenesis-FP, Terpinene3-mutagenesis-RP</td>
 +
<td>KOD-Plus</td>
 +
</tr>
 +
</table>
 +
</div>
 +
(TOYOBO, KOD-Plus-Mutagenesis Kit)
 +
Started from protocol No.2.
<br>
<br>
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<br><br>
 
<br>
<br>
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</p>
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<br>a
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<h3>August 20, 2014</h3>
-
<br>a
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<p class="sentence">
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<br>a
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Restarted mutagenesis from protocol No.3 to obtain plasmid (muta-CuMTS3 in pGEX-6P-2)
 +
<br>
 +
<strong>Transformation</strong>
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th colspan="4">Sample</th>
 +
</tr>
 +
<tr>
 +
<th>Host</th>
 +
<th>Vector</th>
 +
<th>Insert</th>
 +
<th>Primer</th>
 +
</tr>
 +
<tr>
 +
<td rowspan="2">DH5α</td>
 +
<td rowspan="2">pGEX6P-2</td>
 +
<td rowspan="2">Mu-ta-CuMTS3</td>
 +
<td>Terpinene3-mutagenesis-FP and</td>
 +
</tr>
 +
<tr>
 +
<td>Terpinene3-mutagenesis-RP</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<br>
 +
<br>
 +
</p>
 +
 
 +
<h3>August 21, 2014</h3>
 +
<p class="sentence">
 +
<strong>Colony Isolation</strong><br>
 +
Isolated each colony from two plates which were inoculated overnight
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th colspan="4">Sample</th>
 +
<th></th>
 +
</tr>
 +
<tr>
 +
<th>Host</th>
 +
<th>Vector</th>
 +
<th>Insert</th>
 +
<th>Primer</th>
 +
<th>Section</th>
 +
</tr>
 +
<tr>
 +
<td>DH5α</td>
 +
<td>pSB1C3</td>
 +
<td>Mu-ta-CuMTS3</td>
 +
<td>Terpinene3-mutagenesis-FP,<br>Terpinene3-mutagenesis-RP</td>
 +
<td>あ-み</td>
 +
</tr>
 +
</table>
 +
</div>
 +
Chose five isolated colonies whose Code Names are あ,か,さ,た andな and mini-preped them.
 +
<br>
 +
<br>
 +
</p>
 +
 
 +
<h3>August 22, 2014</h3>
 +
<p class="sentence">
 +
<strong>Restriction Digest</strong>
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th>Sample</th>
 +
<th></th>
 +
<th></th>
 +
</tr>
 +
<tr>
 +
<th>Host</th>
 +
<th>Vector</th>
 +
<th>Insert</th>
 +
<th>Enzyme</th>
 +
<th>Buffer</th>
 +
</tr>
 +
<tr>
 +
<td>DH5α  あ</td>
 +
<td>pGEX6P-2</td>
 +
<td>Mu-ta-CuMTS3</td>
 +
<td>Sal1</td>
 +
<td>Fast Di-gest buffer</td>
 +
</tr>
 +
<tr>
 +
<td>DH5α  か</td>
 +
<td>pGEX6P-2</td>
 +
<td>Mu-ta-CuMTS3</td>
 +
<td>Sal1</td>
 +
<td>Fast Di-gest buffer</td>
 +
</tr>
 +
<tr>
 +
<td>DH5α  さ</td>
 +
<td>pGEX6P-2</td>
 +
<td>Mu-ta-CuMTS3</td>
 +
<td>Sal1</td>
 +
<td>Fast Di-gest buffer</td>
 +
</tr>
 +
<tr>
 +
<td>DH5α  た</td>
 +
<td>pGEX6P-2</td>
 +
<td>Mu-ta-CuMTS3</td>
 +
<td>Sal1</td>
 +
<td>Fast Di-gest buffer</td>
 +
</tr>
 +
<tr>
 +
</table>
 +
</div>
 +
<br>
 +
<strong>AGE</strong>
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th></th>
 +
<th colspan="4">Sample</th>
 +
<th></th>
 +
</tr>
 +
<tr>
 +
<th>Lane</th>
 +
<th>Marker</th>
 +
<th>Host</th>
 +
<th>Vector</th>
 +
<th>Insert</th>
 +
<th>Enzyme</th>
 +
</tr>
 +
<tr>
 +
<td>1</td>
 +
<td>1kb Ladder</td>
 +
<td></td>
 +
<td></td>
 +
<td></td>
 +
<td></td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<td></td>
 +
<td>DH5α  あ</td>
 +
<td>pGEX6P-2</td>
 +
<td>Muta-CuMTS3</td>
 +
<td>Sal1</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<td></td>
 +
<td>DH5α  か</td>
 +
<td>pGEX6P-2</td>
 +
<td>Muta-CuMTS3</td>
 +
<td>Sal1</td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<td></td>
 +
<td>DH5α  さ</td>
 +
<td>pGEX6P-2</td>
 +
<td>Muta-CuMTS3</td>
 +
<td>Sal1</td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<td></td>
 +
<td>DH5α  た</td>
 +
<td>pGEX6P-2</td>
 +
<td>Muta-CuMTS3</td>
 +
<td>Sal1</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<br>
 +
<strong>Restriction Digest</strong>
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th colspan="3">Sample</th>
 +
<th></th>
 +
<th></th>
 +
</tr>
 +
<tr>
 +
<th>Host</th>
 +
<th>Vector</th>
 +
<th>Insert</th>
 +
<th>Enzyme</th>
 +
<th>Buffer</th>
 +
</tr>
 +
<tr>
 +
<td>DH5α  か</td>
 +
<td>pGEX6P-2</td>
 +
<td>CuMTS3</td>
 +
<td>EcoR1</td>
 +
<td>Fast Di-gest buffer</td>
 +
</tr>
 +
<tr>
 +
<td>DH5α  か</td>
 +
<td>pGEX6P-2</td>
 +
<td>Mu-ta-CuMTS3</td>
 +
<td>EcoR1</td>
 +
<td>Fast Di-gest buffer</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<br>
 +
<strong>AGE</strong>
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th rowlspan="2">Sample</th>
 +
<th colspan="4">Sample</th>
 +
<th></th>
 +
</tr>
 +
<tr>
 +
<th>Marker</th>
 +
<th>Host</th>
 +
<th>Vector</th>
 +
<th>Insert</th>
 +
<th>Enzyme</th>
 +
</tr>
 +
<tr>
 +
<td>1</td>
 +
<td>1kb Ladder</td>
 +
<td></td>
 +
<td></td>
 +
<td></td>
 +
<td></td>
 +
</tr>
 +
<tr>
 +
<td>2</td>
 +
<td>DH5α  か</td>
 +
<td>pGEX6P-2</td>
 +
<td>CuMTS3</td>
 +
<td>EcoR1</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<td>DH5α  か</td>
 +
<td>pGEX6P-2</td>
 +
<td>Mu-ta-CuMTS3</td>
 +
<td>EcoR1</td>
 +
</tr>
 +
</table>
 +
</div>
 +
An illegal EcoRⅠrestriction site was removed.<br>
 +
<br>
 +
<strong>PCR</strong>
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th colspan="3">Sample</th>
 +
<th></th>
 +
</tr>
 +
<tr>
 +
<th>Vector</th>
 +
<th>Insert</th>
 +
<th>Primer</th>
 +
<th>DNA polymerase</th>
 +
</tr>
 +
<tr>
 +
<td>か  pGEX6P-2</td>
 +
<td>mu-ta-CuMTS3</td>
 +
<td>terpinene3-prefix and ter-pinene3-suffix</td>
 +
<td>KOD-FX-Neo-Plus</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<br>
 +
<br>
 +
</p>
 +
 
 +
<h3>August 30, 2014</h3>
 +
<strong>PCR</strong>
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th colspan="3">Sample</th>
 +
<th></th>
 +
</tr>
 +
<tr>
 +
<th>Vector</th>
 +
<th>Insert</th>
 +
<th>Primer</th>
 +
<th>DNA polymerase</th>
 +
</tr>
 +
<tr>
 +
<td>か  pGEX6P-2</td>
 +
<td>mu-ta-CuMTS3</td>
 +
<td>terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1</td>
 +
<td>Taq</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<br>
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th></th>
 +
<th colspan="4">Sample</th>
 +
</tr>
 +
<tr>
 +
<th>Lane</th>
 +
<th>Marker</th>
 +
<th>Vector</th>
 +
<th>Insert</th>
 +
<th>Primer</th>
 +
</tr>
 +
<tr>
 +
<td>1</td>
 +
<td>1kb Ladder</td>
 +
<td></td>
 +
<td></td>
 +
<td></td>
 +
</tr>
 +
<tr>
 +
<td>1</td>
 +
<td>1kb Ladder</td>
 +
<td></td>
 +
<td></td>
 +
<td></td>
 +
</tr>
 +
<tr>
 +
<td>1</td>
 +
<td>1kb Ladder</td>
 +
<td></td>
 +
<td></td>
 +
<td></td>
 +
</tr>
 +
<tr>
 +
<td>1</td>
 +
<td>1kb Ladder</td>
 +
<td></td>
 +
<td></td>
 +
<td></td>
 +
</tr>
 +
</table>
 +
</div>
<br>
<br>
-
<br><br>
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</tr>
<br>
<br>
 +
</p>
 +
 +
<h3>
 +
<td></td>
 +
<td></td>
 +
<td></td>
 +
<td></td>
 +
</tr>
<br>a
<br>a
<br>a
<br>a

Revision as of 16:37, 15 October 2014

LabNote

June

June 10, 2014

OKAMOTO
Miniprep

Host Sample
Vector Insert
BL21(DE3) pGEX6P-2 CuMTSE1
BL21(DE3) pGEX6P-2 CuMTSE2
BL21(DE3) pGEX6P-2 CuMTS3
BL21(DE3) pGEX6P-2 CuMTS62

Restriction Digest
Host Sample Enzyme Buffer
Vector Insert
BL21(DE3) pGEX6P-2 CuMTSE1 EcoR1 Buffer H
BL21(DE3) pGEX6P-2 CuMTSE2 EcoR1 Buffer H
BL21(DE3) pGEX6P-2 CuMTS3 EcoR1 Buffer H
BL21(DE3) pGEX6P-2 CuMTS62 EcoR1 Buffer H
BL21(DE3) pGEX6P-2 CuMTSE1 Sal1, Not1 Buffer H
BL21(DE3) pGEX6P-2 CuMTSE2 Sal1, Not1 Buffer H
BL21(DE3) pGEX6P-2 CuMTS3 Sal1, Not1 Buffer H
BL21(DE3) pGEX6P-2 CuMTS62 Sal1, Not1 Buffer H


June 11, 2014

KOBAYASHI
AGE

Lane Sample Enzyme
Marker Vector Insert
1 λDNA-HindⅢ
2 pGEX6P-2 CuMTSE1 N.D
3 pGEX6P-2 CuMTSE1 EcoR1
4 pGEX6P-2 CuMTSE1 Sal1, Not1
5 pGEX6P-2 CuMTSE2 N.D
6 pGEX6P-2 CuMTSE2 EcoR1
7 pGEX6P-2 CuMTSE2 Sal1, Not1
8 pGEX6P-2 CuMTS3 N.D
9 pGEX6P-2 CuMTS3 EcoR1
10 pGEX6P-2 CuMTS3 Sal1, Not1
11 pGEX6P-2 CuMTS62 N.D
12 pGEX6P-2 CuMTS62 EcoR1
13 pGEX6P-2 CuMTS62 Sal1, Not1


June 12, 2014

KOBAYASHI
Transformation

Host Sample
Vector Insert
BL21(DE3)pLysS pGEX6P-2 CuMTSE1
BL21(DE3)pLysS pGEX6P-2 CuMTSE2
BL21(DE3)pLysS pGEX6P-2 CuMTS3
BL21(DE3)pLysS pGEX6P-2 CuMTS62


June 13, 2014

KOBAYASHI
Colony Isolation
Select 6 colonies per plate from 4 plates from June 12.

Host Sample Section Enzyme
Vector Insert
BL21(DE3)pLysS pGEX6P-2 CuMTSE1 1-a, 1-b
BL21(DE3)pLysS pGEX6P-2 CuMTSE2 2-a, 2-b
BL21(DE3)pLysS pGEX6P-2 CuMTS3 3-a, 3-b
BL21(DE3)pLysS pGEX6P-2 CuMTS62 6-a, 6-b


June 14, 2014

ONISHI
Miniprep

Host Sample
Vector Insert
1-a pGEX6P-2 CuMTSE1
1-b pGEX6P-2 CuMTSE1
2-a pGEX6P-2 CuMTSE2
2-b pGEX6P-2 CuMTSE2
3-a pGEX6P-2 CuMTS3
3-b pGEX6P-2 CuMTS3
6-a pGEX6P-2 CuMTS62
6-b pGEX6P-2 CuMTS62

Miniprep
Host Sample
Vector Insert
BL21(DE3) pGEX6P-2 CuMTSE1
BL21(DE3) pGEX6P-2 CuMTSE2
BL21(DE3) pGEX6P-2 CuMTS3
BL21(DE3) pGEX6P-2 CuMTS62

Restriction Digest
Host Sample Enzyme Buffer
Vector Insert
1-a pGEX6P-2 CuMTSE1 EcoR1 Buffer H
1-b pGEX6P-2 CuMTSE1 EcoR1 Buffer H
2-a pGEX6P-2 CuMTSE2 EcoR1 Buffer H
2-b pGEX6P-2 CuMTSE2 EcoR1 Buffer H
3-a pGEX6P-2 CuMTS3 EcoR1 Buffer H
3-b pGEX6P-2 CuMTS3 EcoR1 Buffer H
6-a pGEX6P-2 CuMTS62 EcoR1 Buffer H
6-b pGEX6P-2 CuMTS62 EcoR1 Buffer H
BL21(DE3) pGEX6P-2 CuMTSE1 EcoR1 Buffer H
BL21(DE3) pGEX6P-2 CuMTSE2 EcoR1 Buffer H
BL21(DE3) pGEX6P-2 CuMTS3 EcoR1 Buffer H
BL21(DE3) pGEX6P-2 CuMTS62 EcoR1 Buffer H

AGE
Lane Sample Insert Enzyme Buffer
Marker Host Vector
1 1kb Ladder
2 λDNA-HindⅢ
3 1-a pGEX6P-2 CuMTSE1 EcoR1 Buffer H
4 1-b pGEX6P-2 CuMTSE1 EcoR1 Buffer H
5 2-a pGEX6P-2 CuMTSE2 EcoR1 Buffer H
6 2-b pGEX6P-2 CuMTSE2 EcoR1 Buffer H
7 3-a pGEX6P-2 CuMTS3 EcoR1 Buffer H
8 3-b pGEX6P-2 CuMTS3 EcoR1 Buffer H
9 6-a pGEX6P-2 CuMTS62 EcoR1 Buffer H
10 6-b pGEX6P-2 CuMTS62 EcoR1 Buffer H
11 BL21(DE3) pGEX6P-2 CuMTSE1 EcoR1 Buffer H
12 BL21(DE3) pGEX6P-2 CuMTSE2 EcoR1 Buffer H
13 BL21(DE3) pGEX6P-2 CuMTS3 EcoR1 Buffer H
14 BL21(DE3) pGEX6P-2 CuMTS62 EcoR1 Buffer H

Applied λDNA-HindⅢ to compare and contrast AGE of July 14 and June 11.


June 16, 2014

YAMAJI
Preculture

Host Sample
Vector Insert
BL21(DE3)pLysS pGEX6P-2 CuMTSE1
BL21(DE3)pLysS pGEX6P-2 CuMTSE2
BL21(DE3)pLysS pGEX6P-2 CuMTS3
BL21(DE3)pLysS pGEX6P-2 CuMTS62
BL21(DE3)pLysS pGEX6P-2

Main Culture
Host Sample IPTG
Vector Insert
BL21(DE3)pLysS pGEX6P-2 CuMTSE1 Induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE2 Induced
BL21(DE3)pLysS pGEX6P-2 CuMTS3 Induced
BL21(DE3)pLysS pGEX6P-2 CuMTS62 Induced
BL21(DE3)pLysS pGEX6P-2 Induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE1 Non-induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE2 Non-induced
BL21(DE3)pLysS pGEX6P-2 CuMTS3 Non-induced
BL21(DE3)pLysS pGEX6P-2 CuMTS62 Non-induced
BL21(DE3)pLysS pGEX6P-2 Non-induced


June 18, 2014

YAMAJI
Preculture

Host Sample
Vector Insert
BL21(DE3)pLysS pGEX6P-2 CuMTSE1
BL21(DE3)pLysS pGEX6P-2 CuMTSE2

Main Culture
Host Sample IPTG
Vector Insert
BL21(DE3)pLysS pGEX6P-2 Induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE1 Induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE2 Induced
BL21(DE3)pLysS pGEX6P-2 Non-induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE1 Non-induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE2 Non-induced

Protein Extraction (E.coli)
Host Sample IPTG
Vector Insert
BL21(DE3)pLysS pGEX6P-2 Induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE1 Induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE2 Induced
BL21(DE3)pLysS pGEX6P-2 Non-induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE1 Non-induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE2 Non-induced

Preparations for SDS-PAGE
Add SDS sample buffer to protein extractions in a flask, incubate at 37℃ for 15 minutes and keep it at 4℃.


June 19, 2014

KITOMI
SDS-PAGE

Host Sample IPTG
Vector Insert
BL21(DE3)pLysS pGEX6P-2 Induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE1 Induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE2 Induced
BL21(DE3)pLysS pGEX6P-2 Non-induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE1 Non-induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE2 Non-induced


June 21, 2014

SOGA
Miniprep

Host Sample
Vector Insert
BL21(DE3)pLysS pGEX6P-2 CuMTSE1
BL21(DE3)pLysS pGEX6P-2 CuMTSE2

PCR
Vector Sample DNA Polymerase
Insert Primer
pGEX6P-2 CuMTSE1 pGEX6P-2F1, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTSE1 pGEX6P-2F2, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTSE2 pGEX6P-2F1, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTSE2 pGEX6P-2F2, pGEX6P-2R KOD-FX-NEO

AGE
Lane Sample Insert Primer
Marker Host Vector
1 1kb Ladder
2 BY4247 p427-TEF
3 pGEX6P-2 CuMTSE1 pGEX6P-2F1, pGEX6P-2R
4 pGEX6P-2 CuMTSE1 pGEX6P-2F2, pGEX6P-2R
5 pGEX6P-2 CuMTSE2 pGEX6P-2F1, pGEX6P-2R
6 pGEX6P-2 CuMTSE2 pGEX6P-2F2, pGEX6P-2R


June 24, 2014

SOGA
Preculture

Host Sample
Vector Insert
BL21(DE3)pLysS
BL21(DE3)pLysS pGEX6P-2 CuMTSE1
BL21(DE3)pLysS pGEX6P-2 CuMTSE2


June 25, 2014

KOBAYASHI

Cultivated following samples in Big Scale at 30℃ overnight in shaken culture.

Host Sample
Vector Insert
BL21(DE3)pLysS
BL21(DE3)pLysS pGEX6P-2 CuMTSE1
BL21(DE3)pLysS pGEX6P-2 CuMTSE2

DNA Refinement1
Sample
DNA Primer
CuMTSE1 pGEX6P-2F1, pGEX6P-2R
CuMTSE1 pGEX6P-2F2, pGEX6P-2R

Restriction Digest
Vector Sample Enzyme Buffer
DNA Primer
CuMTSE1 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1 Buffer H
CuMTSE1 pGEX6P-2F2, pGEX6P-2R Spe1, Xho1 Buffer H
p427-TEF Spe1, Xho1 Buffer H

DNA Refinement2
Vector Sample Enzyme
DNA Primer
CuMTSE1 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
CuMTSE1 pGEX6P-2F2, pGEX6P-2R Spe1, Xho1
p427-TEF Spe1, Xho1

Main Culture
Host Sample IPTG
Vector Insert
BL21(DE3)pLysS pGEX6P-2 Induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE1 Induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE2 Induced
BL21(DE3)pLysS pGEX6P-2 Non-induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE1 Non-induced
BL21(DE3)pLysS pGEX6P-2 CuMTSE2 Non-induced

Ligation
Sample
Vector DNA Primer
p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R
p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R
p427-TEF CuMTSE2 pGEX6P-2F1, pGEX6P-2R
p427-TEF CuMTSE2 pGEX6P-2F2, pGEX6P-2R
DNA ligase: DNA Ligation Kit ‹Mighty Mix› (TAKARA BIO INC.)

Transformation
Host Sample
Vector Insert Primer
DH5α p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R
DH5α p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R
DH5α p427-TEF CuMTSE2 pGEX6P-2F1, pGEX6P-2R
DH5α p427-TEF CuMTSE2 pGEX6P-2F2, pGEX6P-2R


June 26, 2014

SAWANO
Colony Isolation
Isolate each colony from the plates which were inoculated on June 25.

Host Sample Section
Vector Insert Primer
DH5α p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R 1～4
DH5α p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R a～h
DH5α p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R ア～タ


June 27, 2014

SHIMADA
Colony Sweep

Lane Sample
Host Vector Insert
1 BL21(DE3)pLysS pGEX6P-2 CuMTSE1
2 1
3 2
4 3
5 4
6 a
7 b
8 c
9 d
10 e
11 f
12 g
13 h

Colony Sweep
Lane Sample
Host Vector Insert
1 BL21(DE3)pLysS pGEX6P-2 CuMTSE1
2 ア
3 イ
4 ウ
5 エ
6 オ
7 カ
8 キ
9 ク
10 ケ
11 コ
12 サ
13 シ
14 ス
15 セ
16 ソ
17 タ

Identified targeted genes in 4, c, h, イ, カ, セ

Miniprep
Sample
Host
4
C
H
イ
カ
セ

Restriction Digest
Sample Enzyme Buffer
Host
4 Spe1, Xho1 Buffer H
C Spe1, Xho1 Buffer H
H Spe1, Xho1 Buffer H
イ Spe1, Xho1 Buffer H
カ Spe1, Xho1 Buffer H

AGE
Lane Sample Primer Enzyme
Marker Host Insert
1 1kb Ladder
2 CuMTSE1 pGEX6P-2F1, pGEX6P-2R
3 4 Spe1, Xho1
4 4 Spe1, Xho1
5 4 N.D
6 CuMTSE1 pGEX6P-2F2, pGEX6P-2R
7 c Spe1, Xho1
8 c Spe1, Xho1
9 c N.D
10 h Spe1, Xho1
11 h Spe1, Xho1
12 h N.D

Transformation (electroportation)

Host Sample
Vector Insert Primer
BY4247 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R
BY4247 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R

June 30, 2014

SOGA
Inoculation
Divide each of 2 plates into 8 sections and inoculate following samples on them.

Host Sample Section
Vector Insert Primer
BY4247 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R A～H
BY4247 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R 一～ハ
Cultivate 17 samples in a small scale at 30℃ for 6 hours in shaken culture.


July

July 1, 2014

KOBAYASHI
Protein Extraction (Saccharomyces cerevisiae)

Host Sample
Vector Insert Primer
BY4247 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R
BY4247 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R


July 2, 2014

ONISHI
Miniprep

Host Sample
Vector Insert Primer
BY4247 p427-TEF
BY4247 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R
BY4247 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R

Restriction Digest
Sample Enzyme Buffer
Vector Insert Primer
p427-TEF CuMTSE1 EcoR1 Buffer H
p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R EcoR1 Buffer H
p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R EcoR1 Buffer H
p427-TEF CuMTSE1 Sac1, Kpn1 Buffer L
p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R Sac1, Kpn1 Buffer L
p427-TEF CuMTSE1 pGEX6P-2F2,pGEX6P-2R Sac1, Kpn1 Buffer L

AGE
Lane Sample Enzyme
Marker Vector Insert Primer
1 p427-TEF N.D
2 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R N.D
3 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R N.D
4 1kb Ladder
5 p427-TEF EcoR1
6 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R EcoR1
7 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R EcoR1
8 1kb Ladder
9 p427-TEF Kpn1
10 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R Kpn1
11 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R Kpn1
12 1kb Ladder
13 p427-TEF Sac1
14 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R Sac1
15 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R Sac1

Restriction Digest
Sample Enzyme Buffer
Vector Insert Primer
p427-TEF CuMTSE1 EcoR1 Buffer H
p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R EcoR1 Buffer H
p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R EcoR1 Buffer H
p427-TEF CuMTSE1 Sac1 Buffer L
p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R Sac1 Buffer L
p427-TEF CuMTSE1 pGEX6P-2F2,pGEX6P-2R Sac1 Buffer L
p427-TEF CuMTSE1 Kpn1 Buffer L
p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R Kpn1 Buffer L
p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R Kpn1 Buffer L
p427-TEF CuMTSE1 Spe1, Xho1 Buffer H
p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1 Buffer H
p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R Spe1, Xho1 Buffer H

AGE
Lane Sample Enzyme
Marker Vector Insert Primer
1 1kb Ladder
2 p427-TEF CuMTSE1 EcoR1
3 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R EcoR1
4 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R EcoR1
5 p427-TEF CuMTSE1 Kpn1
6 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R Kpn1
7 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R Kpn1
8 p427-TEF CuMTSE1 Sac1
9 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R Sac1
10 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R Sac1
11 p427-TEF CuMTSE1 Spe1, Xho1
12 p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
13 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R Spe1, Xho1


July 3, 2014

OKAMOTO
PCR

Sample DNA Polymerase
Vector Insert Primer
pGEX6P-2 CuMTSE2 pGEX6P-2F1, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTSE2 pGEX6P-2F2, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTS3 pGEX6P-2F1, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTS3 pGEX6P-2F2, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTS62 pGEX6P-2F1, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTS62 pGEX6P-2F2, pGEX6P-2R KOD-FX-NEO

Protein Extraction (Saccharomyces cerevisiae)
Host Sample
Vector Insert Primer
BY4247 p427-TEF
A p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R
六 p427-TEF CuMTSE1 pGEX6P-2F2, pGEX6P-2R


July 4, 2014

SHIMADA
AGE

Lane Sample/Marker
Marker Vector Insert Primer
1 1kb Ladder
2 pGEX6P-2 CuMTSE2 pGEX6P-2F1, pGEX6P-2R
3 pGEX6P-2 CuMTSE2 pGEX6P-2F2, pGEX6P-2R
4 pGEX6P-2 CuMTS3 pGEX6P-2F1, pGEX6P-2R
5 pGEX6P-2 CuMTS3 pGEX6P-2F2, pGEX6P-2R
6 pGEX6P-2 CuMTS62 pGEX6P-2F1, pGEX6P-2R
7 pGEX6P-2 CuMTS62 pGEX6P-2F2, pGEX6P-2R

PCR
Sample DNA Polymerase
Vector Insert Primer
pGEX6P-2 CuMTSE2 pGEX6P-2F1, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTSE2 pGEX6P-2F2, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTS3 pGEX6P-2F1, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTS3 pGEX6P-2F2, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTS62 pGEX6P-2F1, pGEX6P-2R KOD-FX-NEO
pGEX6P-2 CuMTS62 pGEX6P-2F2, pGEX6P-2R KOD-FX-NEO

AGE
Lane Sample
Marker Vector Insert Primer
1 1kb Ladder
2 pGEX6P-2 CuMTSE2 pGEX6P-2F1, pGEX6P-2R
3 pGEX6P-2 1kb Ladder+K83:N91 pGEX6P-2F2, pGEX6P-2R
4 pGEX6P-2 CuMTS3 pGEX6P-2F1, pGEX6P-2R
5 pGEX6P-2 CuMTS3 pGEX6P-2F2, pGEX6P-2R
6 pGEX6P-2 CuMTS62 pGEX6P-2F1, pGEX6P-2R
7 pGEX6P-2 CuMTS62 pGEX6P-2F2, pGEX6P-2R


July 5, 2014

KOBAYASHI
SDS-PAGE

Host Sample
Vector Insert Primer
BL21(DE3)pLysS pGEX6P-2
4 pGEX6P-2 CuMTSE1 pGEX6P-2F1, pGEX6P-2R
C pGEX6P-2 CuMTSE2 pGEX6P-2F2, pGEX6P-2R
BY4247 p427-TEF
A p427-TEF CuMTSE1 pGEX6P-2F1, pGEX6P-2R
六 p427-TEF CuMTSE2 pGEX6P-2F2, pGEX6P-2R


July 6, 2014

SOGA
DNA Refinement1

Sample
DNA Primer
CuMTSE2 pGEX6P-2F1, pGEX6P-2R
CuMTSE2 pGEX6P-2F2, pGEX6P-2R
CuMTS3 pGEX6P-2F1, pGEX6P-2R
CuMTS62 pGEX6P-2F1, pGEX6P-2R

Restriction Digest
Vector Sample Enzyme Buffer
DNA Primer
CuMTSE2 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1 Fast digest buffer
CuMTSE2 pGEX6P-2F2, pGEX6P-2R Spe1, Xho1 Fast digest buffer
CuMTS3 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1 Fast digest buffer
CuMTS62 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1 Fast digest buffer
p427-TEF Spe1, Xho1 Fast digest buffer

DNA Refinement2
Sample Enzyme
DNA Primer
CuMTSE2 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
CuMTSE2 pGEX6P-2F2, pGEX6P-2R Spe1, Xho1

Ligation
Vector Sample Enzyme
DNA Primer
p427-TEF CuMTSE2 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
p427-TEF CuMTSE2 pGEX6P-2F2, pGEX6P-2R Spe1, Xho1

DNA ligase: DNA Ligation Kit ‹Mighty Mix› (TAKARA BIO INC.)

Transformation (E.coli)
Host Sample Enzyme
Vector Insert Primer
DH5α p427-TEF CuMTSE2 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
DH5α p427-TEF CuMTSE2 pGEX6P-2F2, pGEX6P-2R Spe1, Xho1
DH5α p427-TEF CuMTS3 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
DH5α p427-TEF CuMTS62 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1



July 7, 2014

KOBAYASHI
Colony Isolation
Isolate each colony from the plates which were inoculated on July 6.

Host Sample Section
Vector Insert Primer
DH5α p427-TEF CuMTSE2 pGEX6P-2F1, pGEX6P-2R 1～16
DH5α p427-TEF CuMTS3 pGEX6P-2F1, pGEX6P-2R ア～エ
DH5α p427-TEF CuMTS62 pGEX6P-2F1, pGEX6P-2R a～p

Transformation (E.coli)
Host Sample Enzyme
Vector Insert Primer
DH5α p427-TEF CuMTSE2 pGEX6P-2F2, pGEX6P-2R Spe1, Xho1


July 8, 2014

OKAMOTO
Colony Sweep

Lane Sample
Host
1 DH5α p427-TEF
2 a
3 b
4 c
5 d
6 e
7 f
8 g
9 h
10 i
11 j
12 k
13 l
14 m
15 n
16 o
17 p

Colony Sweep
Lane Sample
Host
1 DH5α p427-TEF
2 ア
3 イ
4 ウ
5 エ

Colony Sweep
Lane Sample
Host
1 DH5α p427-TEF
2 1
3 2
4 3
5 4
6 5
7 6
8 7
9 8
10 9
11 10
12 11
13 12
14 13
15 14
16 15
17 16


July 9, 2014

OKAMOTO
Main Culture

Sample
Host
d
h
l
ア
イ
エ

Mini Prep
Sample
Host
D
H
L
ア
イ
エ

Restriction Digest
Vector Sample Enzyme Buffer
Insert Primer
p427-TEF(d) CuMTS3 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1 Fast digest buffer
p427-TEF(h) CuMTS3 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1 Fast digest buffer
p427-TEF(l) CuMTS3 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1 Fast digest buffer
p427-TEF(ア) CuMTS62 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1 Fast digest buffer
p427-TEF(イ) CuMTS62 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1 Fast digest buffer
p427-TEF(エ) CuMTS62 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1 Fast digest buffer

AGE
Lane Sample Enzyme
Vector Insert Primer
1 p427-TEF(ア) CuMTS3 pGEX6P-2F1, pGEX6P-2R N.D
2 p427-TEF(ア) CuMTS3 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
3 p427-TEF(イ) CuMTS3 pGEX6P-2F1, pGEX6P-2R N.D
4 p427-TEF(イ) CuMTS3 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
5 p427-TEF(エ) CuMTS3 pGEX6P-2F1, pGEX6P-2R N.D
6 p427-TEF(エ) CuMTS3 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
7 p427-TEF(d) CuMTS62 pGEX6P-2F1, pGEX6P-2R N.D
8 p427-TEF(d) CuMTS62 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
9 p427-TEF(h) CuMTS62 pGEX6P-2F1, pGEX6P-2R N.D
10 p427-TEF(h) CuMTS62 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
11 p427-TEF(l) CuMTS62 pGEX6P-2F1, pGEX6P-2R N.D
12 p427-TEF(l) CuMTS62 pGEX6P-2F1, pGEX6P-2R Spe1, Xho1
Marker: 1kb Ladder

August

August 19, 2014

Conducted mutagenesis to remove an illegal restriction site (EcoR1)
InversePCR

Sample
Vector Insert Primer DNA polymer-ase
pGEX6P-2 CuMTS3 Ter-pinene3-mutagenesis-FP, Terpinene3-mutagenesis-RP KOD-Plus
(TOYOBO, KOD-Plus-Mutagenesis Kit) Started from protocol No.2.

August 20, 2014

Restarted mutagenesis from protocol No.3 to obtain plasmid (muta-CuMTS3 in pGEX-6P-2)
Transformation

Sample
Host Vector Insert Primer
DH5α pGEX6P-2 Mu-ta-CuMTS3 Terpinene3-mutagenesis-FP and
Terpinene3-mutagenesis-RP


August 21, 2014

Colony Isolation
Isolated each colony from two plates which were inoculated overnight

Sample
Host Vector Insert Primer Section
DH5α pSB1C3 Mu-ta-CuMTS3 Terpinene3-mutagenesis-FP,
Terpinene3-mutagenesis-RP		 あ-み
Chose five isolated colonies whose Code Names are あ,か,さ,た andな and mini-preped them.

August 22, 2014

Restriction Digest

Sample
Host Vector Insert Enzyme Buffer
DH5α あ pGEX6P-2 Mu-ta-CuMTS3 Sal1 Fast Di-gest buffer
DH5α か pGEX6P-2 Mu-ta-CuMTS3 Sal1 Fast Di-gest buffer
DH5α さ pGEX6P-2 Mu-ta-CuMTS3 Sal1 Fast Di-gest buffer
DH5α た pGEX6P-2 Mu-ta-CuMTS3 Sal1 Fast Di-gest buffer

AGE
Sample
Lane Marker Host Vector Insert Enzyme
1 1kb Ladder
2 DH5α あ pGEX6P-2 Muta-CuMTS3 Sal1
3 DH5α か pGEX6P-2 Muta-CuMTS3 Sal1
2 DH5α さ pGEX6P-2 Muta-CuMTS3 Sal1
2 DH5α た pGEX6P-2 Muta-CuMTS3 Sal1

Restriction Digest
Sample
Host Vector Insert Enzyme Buffer
DH5α か pGEX6P-2 CuMTS3 EcoR1 Fast Di-gest buffer
DH5α か pGEX6P-2 Mu-ta-CuMTS3 EcoR1 Fast Di-gest buffer

AGE
Sample Sample
Marker Host Vector Insert Enzyme
1 1kb Ladder
2 DH5α か pGEX6P-2 CuMTS3 EcoR1
3 DH5α か pGEX6P-2 Mu-ta-CuMTS3 EcoR1
An illegal EcoRⅠrestriction site was removed.

PCR
Sample
Vector Insert Primer DNA polymerase
か pGEX6P-2 mu-ta-CuMTS3 terpinene3-prefix and ter-pinene3-suffix KOD-FX-Neo-Plus


August 30, 2014

PCR
Sample
Vector Insert Primer DNA polymerase
か pGEX6P-2 mu-ta-CuMTS3 terpinene3-prefix-Xba1 and terpinene3-suffix-Spe1 Taq

Sample
Lane Marker Vector Insert Primer
1 1kb Ladder
1 1kb Ladder
1 1kb Ladder
1 1kb Ladder



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Retrieved from "http://2014.igem.org/Team:KIT-Kyoto/Notebook/Labnote"