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Team:Bielefeld-CeBiTec/Notebook/Journal/Biosafety/Jul
From 2014.igem.org
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| - | Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of <i>dadX</i> | + | 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> |
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| - | Transformation of the oligonucleotide containing the flanking sites for the deletion of <i>dadX</i>. The successful replacement of the | + | 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. |
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| + | </ul> | ||
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| + | <ul> | ||
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| + | 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>) | <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>) | ||
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| + | 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> | ||
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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.
