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Team:Bielefeld-CeBiTec/Notebook/Journal/Biosafety/Jul
From 2014.igem.org
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| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 1 07/07 - 07/13</a></h6> |
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| + | <ul> | ||
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| + | Transformation of the single deletions strains <i>E. coli</i> strains KRX ∆<i>alr</i> and DH5alpha ∆<i>alr</i> with the plasmid pRedET containg the Recombinase using the <a href="http://www.genebridges.com/storage/Manuals_PDF/K006%20Ecoli%20Gene%20Deletion%20Kit-version2.3-2012.pdf" target="_blank">Genebridge RedET-System protocol</a>. | ||
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<div class="tab" id="Week2"> | <div class="tab" id="Week2"> | ||
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| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 2 07/14 - 07/20</a></h6> |
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| - | <div class="content"> | + | <div class="content" style="margin-right:10%; margin-left:10%"> |
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| + | <ul> | ||
| + | <li> | ||
| + | Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of <i>dadX</i> using the primer <a href="http://parts.igem.org/Part:BBa_K1465405" target="_blank">BBa_K1465405</a> and <a href="http://parts.igem.org/Part:BBa_K1465406" target="_blank">BBa_K1465406</a> | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | <ul> | ||
| + | <li> | ||
| + | Purification using the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugationgel" target="_blank">gel extraction clean-up kit</a> from Promega. | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | <ul> | ||
| + | <li> | ||
| + | Transformation of the oligonucleotide containing the flanking sites for the deletion of <i>dadX</i>. The successful replacement of the catabolic alanine racemase was verified via kanamycin selection and | ||
| + | |||
| + | <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dadX_Ec_control1" target="_blank">dadX_Ec_control1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dadX_Ec_control2" target="_blank">dadX_Ec_control2</a>) | ||
| + | |||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands as expected (3004 bp)</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | <li> | ||
| + | Resulting in the genotype DH5aplha ∆<i>alr</i> <i>kan:dadX</i>, while the dadX deletion in the KRX strain was not successful. | ||
| + | </li> | ||
| + | </ul> | ||
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| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 3 07/21 - 07/27</a></h6> |
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| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 4 07/28 - 08/03</a></h6> |
</div> | </div> | ||
| - | <div class="content"> | + | <div class="content" style="margin-right:10%; margin-left:10%"> |
| + | |||
| + | <ul> | ||
| + | <li> | ||
| + | Transformation of the oligonucleotide containing the flanking sites for the deletion of <i>dadX</i> into the KRX ∆<i>alr</i>. The successful replacement of the katabolic alanine racemase was verified via kanamycin selection and | ||
| + | |||
| + | <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dadX_Ec_control1" target="_blank">dadX_Ec_control1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dadX_Ec_control2" target="_blank">dadX_Ec_control2</a>) | ||
| + | |||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands as expected (3004 bp)</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | <li> | ||
| + | Resulting in the genotype KRX ∆<i>alr</i> <i>kan:dadX</i>. | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
</div> | </div> | ||
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Latest revision as of 02:34, 9 October 2014
 |
July |
 |
- Transformation of the single deletions strains E. coli strains KRX ∆alr and DH5alpha ∆alr with the plasmid pRedET containg the Recombinase using the Genebridge RedET-System protocol.
- Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of dadX using the primer BBa_K1465405 and BBa_K1465406
- Purification using the gel extraction clean-up kit from Promega.
-
Transformation of the oligonucleotide containing the flanking sites for the deletion of dadX. The successful replacement of the catabolic alanine racemase was verified via kanamycin selection and
Colony PCR (dadX_Ec_control1, dadX_Ec_control2)
- Annealing temperature: 55 °C
- Bands as expected (3004 bp)
- Resulting in the genotype DH5aplha ∆alr kan:dadX, while the dadX deletion in the KRX strain was not successful.
-
Transformation of the oligonucleotide containing the flanking sites for the deletion of dadX into the KRX ∆alr. The successful replacement of the katabolic alanine racemase was verified via kanamycin selection and
Colony PCR (dadX_Ec_control1, dadX_Ec_control2)
- Annealing temperature: 55 °C
- Bands as expected (3004 bp)
- Resulting in the genotype KRX ∆alr kan:dadX.
