Team:TMU-Tokyo/Notebook experiment/3rd

From 2013.igem.org

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PCR following fragment. (2nd PCR)<Br>
PCR following fragment. (2nd PCR)<Br>
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<Table cellpadding="5">
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<Tr>
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<Td>Fragment</Td><Td>fimE(1N2)</Td><Td>fimE(2N2)</Td><Td>fimE(1N9)</Td><Td>fimE(2N9)</Td>
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</Tr>
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<Tr>
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<Td>Primer</Td><Td>pBAD-N2<Br>fimE-1</Td><Td>pBAD-N2<Br>fimE-2</Td><Td>pBAD-N9<Br>fimE-1</Td><Td>pBAD-N9<Br>fimE-2</Td>
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</Tr>
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<Tr>
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<Td>Templete</Td><Td>pBAD<Br>fimE</Td><Td>pBAD<Br>fimE</Td><Td>pBAD<Br>fimE</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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Result.<Br>
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<IMG Src="https://static.igem.org/mediawiki/2013/2/28/TMU-Tokyo_0919_1.jpg"><Br>
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1-3: 1N2, 4-6: 2N2, 7-9: 1N9, 10-12: 2N9<Br>
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Electro Poration<Br>
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<Table><Tr><Td>Fragment 1, 2, 3 - Ap to <i>Escherichia coli</i> MG1655 red.</Td></Tr></Table>
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Mini prep following plasmid.
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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/2013/5/50/TMU-Tokyo_0919_2.jpg"><Br>
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1: pSB1A3, 2: pSB1C3<Br>
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<Br>
-
 
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Digestion<Br>
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<Table><Tr><Td>fimE, pSB1C3-Km, pSB1A3</Td></Tr></Table>
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Result<Br>
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<IMG Src="https://static.igem.org/mediawiki/2013/2/2f/TMU-Tokyo_0919_3.jpg"><Br>
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1: fimE, 2: pSB1C2-Km, 3: pSB1A3<Br>
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Revision as of 21:10, 27 September 2013



Experiment > 3rd week


September 15th
PCR result

1: Fragment 1, 2: Fragment 2, 3: Fragment 3 - Ap,
4: Fragment 3- Cm, 5: pBAD-fimE


Ligation fragment and vector.
Plasmid nameFragment 1-A+BFragment 2Fragment 3 - Ap
FragmentFragment 1-A
Fragment 1-B		Fragment 2Fragment 3 - Ap
VectorpSB1C3pSB1C3pSB1C3


Transformation following plasmid to Escherichia coli MG1655 red.
Fragment 1, 2, 3 - Ap


September 16th
Transformation result.



Mini prep following plasmid.
Fragment 1, 2, 3 - Ap, 3 - Cm, Km-Blunt
Result

1: Fragment 1, 2: Fragment 3 - Ap,
3-5: Fragment 3 - Cm, 6-8: Km-Blunt


PCR following fragment.
FragmentFragment 1Fragment 2Fragment 3 - ApFragment 3 - Cm
PrimerlacA-IRR/IRL
cynR-SaApR		araCHC
araA-TcC		lacI-R(FRT)2
mhpA-SaApR		p(SaApR)-lacIN
mhpA-CmC6
TempleteIF2
FS2		Fragment 2-C
Fragment 2-D		pSG76A2lacI
FRT-Cm2


September 17th
PCR result.

1-2: Fragment 1, 3-4: Fragment 2,
5-6: Fragment 3 - Cm, 8-10: Fragment 3 - Ap


Electro Poration
Fragment 1, 2, 3 - AP to Escherichia coli MG1655 red.


PCR following fragment.
FragmentFragment 1Fragment 2Fragment 3 - ApFragment 3 - Cm
PrimerlacA-IRR/IRL
cynR-SaApR		araCHC
araA-TcC		lacI-R(FRT)2
mhpA-SaApR		p(SaApR)-lacIN
mhpA-CmC6
TempleteIF2
FS2		Fragment 2-C
Fragment 2-D		pSG76A2lacI
FRT-Cm2


September 18th
PCR result.

1-3: Fragment 1, 4-6: Fragment 2,
7-9: Fragment 3 - Ap, 1-3: Fragment 3 - Cm


Mini prep following plasmid
pSB1C3-Km, pSB1A3


PCR following fragment.
FragmentfimEFragment 1Fragment 2Fragment 3 - Cm
PrimerpBAD-N9
fimE-C-EcoRⅠ		lacA-IRR/IRL
cynR-SaApR		araCHC
araA-TcC		R(SaApR)-lacIN
mhpA-CmC6
TempletepBAD
fimE		IF2
FS2		Fragment 2-C
Fragment 2-D		lacI
FRT-Cm


September 19th
PCR result.

1: fimE, 2-3: Fragment 1, 4-5: Fragment 2,
6-7: Fragment 3 - Cm


PCR following fragment. (2nd PCR)
FragmentfimE(1N2)fimE(2N2)fimE(1N9)fimE(2N9)
PrimerpBAD-N2
fimE-1		pBAD-N2
fimE-2		pBAD-N9
fimE-1		pBAD-N9
fimE-2
TempletepBAD
fimE		pBAD
fimE		pBAD
fimE		pBAD
fimE
Result.

1-3: 1N2, 4-6: 2N2, 7-9: 1N9, 10-12: 2N9


Electro Poration
Fragment 1, 2, 3 - Ap to Escherichia coli MG1655 red.


Mini prep following plasmid.
pSB1A3, pSB1C3
Result

1: pSB1A3, 2: pSB1C3


Digestion
fimE, pSB1C3-Km, pSB1A3
Result

1: fimE, 2: pSB1C2-Km, 3: pSB1A3


September 20th
September 21th
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