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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun
From 2014.igem.org
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<li><b><i>alsS</i></b></li> | <li><b><i>alsS</i></b></li> | ||
<ul> | <ul> | ||
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<li>Glycerin stock created: <i>E. coli</i> KRX pSB1C3-<i>ilvD</i></li> | <li>Glycerin stock created: <i>E. coli</i> KRX pSB1C3-<i>ilvD</i></li> | ||
</ul> | </ul> | ||
| + | </ul> | ||
| + | <li><b><i>kivD</i></b></li> | ||
| + | <ul> | ||
| + | <li>This week we aim to transform the construct from the parts distribution.</li> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li> | ||
| + | <ul> | ||
| + | <li>K538742: pSB1C3-<i>kivD</i> (parts distribution from 2013, plate 1, 24L)</li> | ||
| + | </ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>kivD</i></li> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sequencing" target="_blank">Sequencing</a> confirmed the identity of pSB1C3-<i>kivD</i>, but not the whole sequence of this part was covered. (<a href="href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF</a> and <a href=href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR</a>)</li> | ||
| + | <li>Glycerin stock created: <i>E. coli</i> KRX pSB1C3-<i>kivD</i></li> | ||
| + | </ul> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
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<ul> | <ul> | ||
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<li><b><i>alsS</i></b></li> | <li><b><i>alsS</i></b></li> | ||
<ul> | <ul> | ||
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_pSB1C3" target="_blank">fw_alsS_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS" target="_blank">rv_ilvC_alsS</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_pSB1C3" target="_blank">fw_alsS_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS" target="_blank">rv_ilvC_alsS</a>)</li> | ||
<ul> | <ul> | ||
| - | <li> | + | <li>Annealing temperature: 55 °C</li> |
<li>Bands not as expected (1,8 kb)</li> | <li>Bands not as expected (1,8 kb)</li> | ||
</ul> | </ul> | ||
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvD_ilvC" target="_blank">rv_ilvD_ilvC</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvD_ilvC" target="_blank">rv_ilvD_ilvC</a>)</li> | ||
<ul> | <ul> | ||
| - | <li> | + | <li>Annealing temperature: 55 °C</li> |
<li>Bands not as expected (1,55 kb)</li> | <li>Bands not as expected (1,55 kb)</li> | ||
</ul> | </ul> | ||
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvC_ilvD" target="_blank">fw_ilvC_ilvD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvC_ilvD" target="_blank">fw_ilvC_ilvD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li> | ||
<ul> | <ul> | ||
| - | <li> | + | <li>Annealing temperature: 55 °C</li> |
<li>Bands not as expected (1,95 kb)</li> | <li>Bands not as expected (1,95 kb)</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <li><b><i>kivD</i></b></li> | ||
| + | <ul> | ||
| + | <li>This week we tried to amplify <i>kivD</i> to use it for the Gibson Assembly.</li> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>)</li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands not as expected (1,75 kb)</li> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a>)</li> | ||
<ul> | <ul> | ||
| - | <li> | + | <li>Annealing temperature: 55 °C</li> |
<li>Bands not as expected (2,2 kb)</li> | <li>Bands not as expected (2,2 kb)</li> | ||
</ul> | </ul> | ||
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</ul> | </ul> | ||
</ul> | </ul> | ||
Revision as of 16:15, 30 August 2014
 |
June |
 |
- alsS
- This week we aim to transform the construct from the parts distribution.
- Transformation with electrocompotetent cells
- K539627: pSB1C3-alsS (parts distribution from 2013, plate 1, 24H)
- Plasmid isolation of alsS
- Sequencing confirmed the identity of pSB1C3-alsS, but not the whole sequence of this part was covered. (VF and VR)
- Glycerin stock created: E. coli KRX pSB1C3-alsS
- ilvC
- This week we aim to transform the construct from the parts distribution.
- Transformation with electrocompotetent cells
- K539621: pSB1C3-ilvC (parts distribution from 2013, plate 1, 24F)
- Plasmid isolation of ilC
- Sequencing confirmed the identity of pSB1C3-ilvC, but not the whole sequence of this part was covered. (VF and VR)
- Glycerin stock created: E. coli KRX pSB1C3-ilvC
- ilvD
- This week we aim to transform the construct from the parts distribution.
- Transformation with electrocompotetent cells
- K539626: pSB1C3-ilvD (parts distribution from 2013, plate 1, 22J)
- Plasmid isolation of ilvD
- Sequencing confirmed the identity of pSB1C3-ilvD, but not the whole sequence of this part was covered. (VF and VR)
- Glycerin stock created: E. coli KRX pSB1C3-ilvD
- kivD
- This week we aim to transform the construct from the parts distribution.
- Transformation with electrocompotetent cells
- K538742: pSB1C3-kivD (parts distribution from 2013, plate 1, 24L)
- Plasmid isolation of kivD
- Sequencing confirmed the identity of pSB1C3-kivD, but not the whole sequence of this part was covered. (VF and VR)
- Glycerin stock created: E. coli KRX pSB1C3-kivD
- alsS
- This week we tried to amplify alsS to use it for the Gibson Assembly.
- PCR amplification (fw_alsS_pSB1C3, rv_ilvC_alsS)
- Annealing temperature: 55 °C
- Bands not as expected (1,8 kb)
- ilvC
- This week we tried to amplify ilvC to use it for the Gibson Assembly.
- PCR amplification (fw_alsS_ilvC, rv_ilvD_ilvC)
- Annealing temperature: 55 °C
- Bands not as expected (1,55 kb)
- ilvD
- This week we tried to amplify ilvD to use it for the Gibson Assembly.
- PCR amplification (fw_ilvC_ilvD, rv_kivD_ilvD)
- Annealing temperature: 55 °C
- Bands not as expected (1,95 kb)
- kivD
- This week we tried to amplify kivD to use it for the Gibson Assembly.
- PCR amplification (fw_ilvD_kivD, rv_pSB1C3_kivD)
- Annealing temperature: 55 °C
- Bands not as expected (1,75 kb)
- Backbone
- This week we tried to amplify the backbone pSB1C3 to use it for the Gibson Assembly.
- PCR amplification (fw_kivD_pSB1C3, rv_alsS_pSB1C3)
- Annealing temperature: 55 °C
- Bands not as expected (2,2 kb)
- PCR conditions need to be optimized due to low product quality and missing products
