Team:TMU-Tokyo/Notebook experiment/2nd

From 2013.igem.org

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<b>September 11th</b><Br>
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<b>September 12th</b><Br>
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Digestion
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<Table><Tr><Td>pSB1A3, pSB1C3</Td></Tr></Table>
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Result<Br>
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<IMG Src="https://static.igem.org/mediawiki/2013/5/56/TMU-Tokyo_0912.jpg"><Br>
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1-2: pSB1A3, 3-6: pSB1C3<Br>
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<Br>
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<Br>
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Ligation following vector and fragment.<Br>
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<div style="text-align:center;">
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<Table cellpadding="5">
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<Tr>
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<Td>Plasmid name</Td><Td>Fragment 1</Td><Td>Fragment 2</Td><Td>Fragment 3 - Ap</Td><Td>Fragment 3 - Cm</Td>
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</Tr>
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<Tr>
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<Td>Vector</Td><Td>pSB1C3</Td><Td>pSB1C3</Td><Td>pSB1C3</Td><Td>pSB1A3</Td>
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</Tr>
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<Tr>
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<Td>Fragment</Td><Td>Fragment 1-C</Td><Td>Fragment 2</Td><Td>FRT-Ap</Td><Td>lacI-FRT-Cm</Td>
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</Tr>
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</Table>
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</div>
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<Br>
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<Br>
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Electro Poration
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<Table><Tr><Td>Fragment 1-C, 2 and FRT-Ap to <i>Escherichia coli</i> MG1655 red</Td></Tr></Table>
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<Br>
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<Br>
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PCR following fragment. (1st PCR)<Br>
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<div style="text-align:center;">
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<Table cellpadding="5">
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<Tr>
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<Td>Fragment</Td><Td>pBAD</Td><Td>fimE</Td>
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</Tr>
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<Tr>
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<Td>Primer</Td><Td>pBAD-N2<Br>pBAD-C3</Td><Td>pBAD-fimE<Br>fimE-C-EcoRⅠ</Td>
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</Tr>
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<Tr>
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<Td>Templete</Td><Td>pBAD-MycHis</Td><Td>fimE (BBa_K137007)</Td>
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</Tr>
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</Table>
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</div>
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<Br>
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<Br>
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<b>September 13th</b><Br>
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Electro Poration Result<Br>
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<IMG Src="https://static.igem.org/mediawiki/2013/2/20/TMU-Tokyo_0912_1.jpg"><Br>
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<IMG Src="https://static.igem.org/mediawiki/2013/2/2b/TMU-Tokyo_0912_2.jpg"><Br>
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<IMG Src="https://static.igem.org/mediawiki/2013/4/42/TMU-Tokyo_0912_3.jpg"><Br>
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<Br>
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<Br>
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Digestion following fragment.
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<Table><Tr><Td>attP, Km-Blunt</Td></Tr></Table>
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Result<Br>
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<IMG Src="https://static.igem.org/mediawiki/2013/5/54/TMU-Tokyo_0912_00.jpg"><Br>
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<Br>
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<Br>
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PCR following fragment.
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<div style="text-align:center;">
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<Table cellpadding="5">
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<Tr>
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<Td>Fragment</Td><Td>Km-Blunt</Td><Td>fimE</Td>
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</Tr>
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<Tr>
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<Td>Primer</Td><Td>Km-BluntN<Br>Km-BluntC</Td>
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</Tr>
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<Tr>
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<Td>Templete</Td><Td>pACYC177</Td>
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</Tr>
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</Table>
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</div>
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<Br>
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<Br>
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<b>September 14th</b><Br>
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PCR following fragment.
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<div style="text-align:center;">
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<Table cellpadding="5">
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<Tr>
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<Td>Fragment</Td><Td>Fragment 1</Td><Td>Fragment 2</Td><Td>Fragment 3 - Ap</Td><Td>Fragment 3 - Cm</Td><Td>pBAD-fimE</Td>
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</Tr>
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<Tr>
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<Td>Primer</Td><Td>lacA-IRR/IRL<Br>cynR-SaApR</Td><Td>araCHC<Br>araA-TcC</Td><Td>lacI-R(FRT)2<Br>mhpA-SaApR</Td><Td>R(SaApR)-lacIN<Br>mhpA-CmC6</Td><Td>pBAD-N2<Br>fimE-C-EcoRⅠ</Td>
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</Tr>
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<Tr>
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<Td>Templete</Td><Td>IF2<Br>FS2</Td><Td>Fragment 2-C<Br>Fragment 2-D</Td><Td>pSG76A2</Td><Td>lacI<Br>FRT-Cm</Td><Td>pBAD<Br>fimE</Td>
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</Tr>
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</Table>
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</div>
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<Br>
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<Br>
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Electro Poration<Br>
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<Table><Tr><Td>Fragment 2 (vector: pSB1C3) to <i>Escherichia coli</i> MG1655 red</Td></Tr></Table>
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<Br>
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<Br>
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Latest revision as of 19:37, 27 September 2013



Experiment > 2nd week


September 8th
PCR following fragment. (2nd PCR)
FragmentFragment IF2Fragment FS2
PrimerCmNR-Not
FLP-R		CmC2
FLP-L
TempletePtetR1
FLP		FLP
Sm
Result

1: Fragment IF2, 2: Fragment FS2


PCR following fragment. (3rd PCR)
FragmentFragment 1
PrimerlacA-IRR/IRL
cynR-SaApR
TempleteIF2
FS2
Result



September 9th
PCR following fragment. (3rd PCR)
FragmentFragment 1-AFragment 1-BFragment 1-CFragment 1-D
PrimerlacA-IRR/IRL
cynR-SaApR		lacA-IRR/IRL
cynR-SaApR		lacA-IRR/IRL
cynR-SaApR		CmC2
CmNR-Not
TempleteIF2
FS		IF
FS2		IF2
FS2		IF2
FS2


September 10th
PCR Result (Fragment 1-A, 1-B, 1-C, 1-D)

1: Fragment 1-A, 2: Fragment 1-B,
3: Fragment 1-C, 4: Fragment 1-D


Plasmid Extract attP (BBa_K617004)


Vector Remodeling with PCR. Insertion Recognition sites (NruⅠ).
FragmentC3attP1･2A3
PrimerpSB1C3-NSP2
pSB1C3-NXE2		attP-NruⅠR
attP-NruⅠL		pSB1C3-NSP2
pSB1C3-NXE2
TempleteattPattPIRR
Result

1: C3, 2: attP1･2, 3: A3


DNA Existract following fragment from gel.
C3, A3, 1-A, 1-B, 1-3


September 11th
Digestion
pSB1C3, pSB1A3
Result

1: C3, 2:A3


Electro Pration
Fragment 1, 2, 3 to Escherichia coli MG1655 red


PCR following vector
FragmentpSB1A3pSB1C3
PrimerpSB1C3-NSP2
pSB1C3-NXE2		pSB1C3-NSP2
pSB1C3-NXE2
TempleteattP
IRR		attP
IRR
Result

1-2: pSB1A3, 3-7: pSB1C3


September 12th
Digestion
pSB1A3, pSB1C3
Result

1-2: pSB1A3, 3-6: pSB1C3


Ligation following vector and fragment.
Plasmid nameFragment 1Fragment 2Fragment 3 - ApFragment 3 - Cm
VectorpSB1C3pSB1C3pSB1C3pSB1A3
FragmentFragment 1-CFragment 2FRT-AplacI-FRT-Cm


Electro Poration
Fragment 1-C, 2 and FRT-Ap to Escherichia coli MG1655 red


PCR following fragment. (1st PCR)
FragmentpBADfimE
PrimerpBAD-N2
pBAD-C3		pBAD-fimE
fimE-C-EcoRⅠ
TempletepBAD-MycHisfimE (BBa_K137007)


September 13th
Electro Poration Result





Digestion following fragment.
attP, Km-Blunt
Result



PCR following fragment.
FragmentKm-BluntfimE
PrimerKm-BluntN
Km-BluntC
TempletepACYC177


September 14th
PCR following fragment.
FragmentFragment 1Fragment 2Fragment 3 - ApFragment 3 - CmpBAD-fimE
PrimerlacA-IRR/IRL
cynR-SaApR		araCHC
araA-TcC		lacI-R(FRT)2
mhpA-SaApR		R(SaApR)-lacIN
mhpA-CmC6		pBAD-N2
fimE-C-EcoRⅠ
TempleteIF2
FS2		Fragment 2-C
Fragment 2-D		pSG76A2lacI
FRT-Cm		pBAD
fimE


Electro Poration
Fragment 2 (vector: pSB1C3) to Escherichia coli MG1655 red


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