Team:TMU-Tokyo/Notebook experiment/3rd

From 2013.igem.org

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Transformation following plasmid.
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Transformation following plasmid to <i>Escherichia coli</i> MG1655 red.
<Table><Tr><Td>Fragment 1, 2, 3 - Ap</Td></Tr></Table>
<Table><Tr><Td>Fragment 1, 2, 3 - Ap</Td></Tr></Table>
<Br>
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<b>September 16th</b><Br>
<b>September 16th</b><Br>
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Transformation result.<Br>
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<IMG Src="https://static.igem.org/mediawiki/2013/5/5b/TMU-Tokyo_0916_1.jpg">
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<IMG Src="https://static.igem.org/mediawiki/2013/1/15/TMU-Tokyo_0916_2.jpg">
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<IMG Src="https://static.igem.org/mediawiki/2013/8/87/TMU-Tokyo_0916_3.jpg"><Br>
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<Br>
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Mini prep following plasmid.
Mini prep following plasmid.
<Table><Tr><Td>Fragment 1, 2, 3 - Ap, 3 - Cm, Km-Blunt</Td></Tr></Table>
<Table><Tr><Td>Fragment 1, 2, 3 - Ap, 3 - Cm, Km-Blunt</Td></Tr></Table>
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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>
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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>p(SaApR)-lacIN<Br>mhpA-CmC6</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-Cm2</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 17th</b><Br>
<b>September 17th</b><Br>
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PCR result.<Br>
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<IMG Src="https://static.igem.org/mediawiki/2013/7/7c/TMU-Tokyo_0917.jpg"><Br>
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1-2: Fragment 1, 3-4: Fragment 2,<Br>
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5-6: Fragment 3 - Cm, 8-10: Fragment 3 - Ap<Br>
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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 1, 2, 3 - 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.<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>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>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>p(SaApR)-lacIN<Br>mhpA-CmC6</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-Cm2</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 18th</b><Br>
<b>September 18th</b><Br>
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PCR result.<Br>
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<IMG Src="https://static.igem.org/mediawiki/2013/f/fc/TMU-Tokyo_0917_1.jpg"><Br>
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1-3: Fragment 1, 4-6: Fragment 2,<Br>
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7-9: Fragment 3 - Ap, 1-3: Fragment 3 - Cm<Br>
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<Br>
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<Br>
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Mini prep following plasmid<Br>
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<Table><Tr><Td>pSB1C3-Km, pSB1A3</Td></Tr></Table>
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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>fimE</Td><Td>Fragment 1</Td><Td>Fragment 2</Td><Td>Fragment 3 - Cm</Td>
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</Tr>
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<Tr>
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<Td>Primer</Td><Td>pBAD-N9<Br>fimE-C-EcoRⅠ</Td><Td>lacA-IRR/IRL<Br>cynR-SaApR</Td><Td>araCHC<Br>araA-TcC</Td><Td>R(SaApR)-lacIN<Br>mhpA-CmC6</Td>
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</Tr>
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<Tr>
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<Td>Templete</Td><Td>pBAD<Br>fimE</Td><Td>IF2<Br>FS2</Td><Td>Fragment 2-C<Br>Fragment 2-D</Td><Td>lacI<Br>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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<b>September 19th</b><Br>
<b>September 19th</b><Br>
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PCR result.<Br>
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<IMG Src="https://static.igem.org/mediawiki/2013/c/c4/TMU-Tokyo_0919.jpg"><Br>
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1: fimE, 2-3: Fragment 1, 4-5: Fragment 2,<Br>
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6-7: Fragment 3 - Cm<Br>
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<Br>
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<Br>
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PCR following fragment. (2nd 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>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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<Br>
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<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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<Br>
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<Br>
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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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Digestion<Br>
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<Table><Tr><Td>fimE-pBAD, 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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<Br>
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<Br>
<b>September 20th</b><Br>
<b>September 20th</b><Br>
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<b>September 21th</b><Br>
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Ligation following vector and fragment.<Br>
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<Table><Tr><Td>fimE-pBAD + pSB1A3</Td></Tr></Table><Br>
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<Br>
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<Br>
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Check 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>(control)</Td><Td>Fragment 1</Td><Td>Fragment 2</Td><Td>Fragment 3 - Ap</Td>
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</Tr>
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<Tr>
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<Td>Primer</Td><Td>3 sets of the others</Td><Td>141-64-1<Br>FLP-R</Td><Td>117-4<Br>pBAD-C3</Td><Td>141-1<Br>SaApR</Td>
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</Tr>
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<Tr>
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<Td>Templete</Td><Td><i>Escherichia coli</i><Br>MG1655 red<Br>(Cell suspention)</Td><Td>(Cell suspention)</Td><Td>(Cell suspention)</Td><Td>(Cell suspention)</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/2013/0/0b/TMU-Tokyo_0920.jpg"><Br>
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1-5: Fragment 1, 6: control(fragment 1),<Br>
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7-10: Fragment 2, 11: control(fragment 2),<Br>
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12-15: Fragment 3, 16: control(fragment 3)<Br>
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<Br>
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<Br>
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<b>September 21th</b><Br>
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Mini prep following plasmid.
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<Table><Tr><Td>Km-Blunt in pSB1C3</Td></Tr></Table>
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Result.<Br>
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<IMG Src="https://static.igem.org/mediawiki/2013/c/cc/TMU-Tokyo_0921.jpg"><Br>
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1-3: Km-Blunt in pSB1C3<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>Fragment 1</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>
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</Tr>
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<Tr>
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<Td>Templete</Td><Td>IF2<Br>FS2</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/c/c8/TMU-Tokyo_0921_1.jpg"><Br>
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1-2: Fragment 1<Br>
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<Br>
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<Br>

Latest revision as of 21:49, 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-pBAD, pSB1C3-Km, pSB1A3
Result

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


September 20th
Ligation following vector and fragment.
fimE-pBAD + pSB1A3



Check PCR.
Fragment(control)Fragment 1Fragment 2Fragment 3 - Ap
Primer3 sets of the others141-64-1
FLP-R		117-4
pBAD-C3		141-1
SaApR
TempleteEscherichia coli
MG1655 red
(Cell suspention)		(Cell suspention)(Cell suspention)(Cell suspention)
Result

1-5: Fragment 1, 6: control(fragment 1),
7-10: Fragment 2, 11: control(fragment 2),
12-15: Fragment 3, 16: control(fragment 3)


September 21th
Mini prep following plasmid.
Km-Blunt in pSB1C3
Result.

1-3: Km-Blunt in pSB1C3


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

1-2: Fragment 1


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