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Team:Bielefeld-CeBiTec/Notebook/Journal/Biosafety/Jun
From 2014.igem.org
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Design of the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Integration_alr" target="_blank">Primer</a> for the integration of the konstitutive alanine racemase <i>alr</i> into the pSB1C3-Backbone. | Design of the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Integration_alr" target="_blank">Primer</a> for the integration of the konstitutive alanine racemase <i>alr</i> into the pSB1C3-Backbone. | ||
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| - | <ul>Transformation of the <i>E. coli</i> strains KRX (<a href="http://www.promega.de/resources/protocols/technical-bulletins/101/single-step-krx-competent-cells-protocol" target="_blank">Promega</a>) and DH5alpha (<a href="http://tools.lifetechnologies.com/content/sfs/manuals/11319019.pdf" target="_blank">Invitrogen</a>) 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>. | + | <ul> |
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| + | Transformation of the <i>E. coli</i> strains KRX (<a href="http://www.promega.de/resources/protocols/technical-bulletins/101/single-step-krx-competent-cells-protocol" target="_blank">Promega</a>) and DH5alpha (<a href="http://tools.lifetechnologies.com/content/sfs/manuals/11319019.pdf" target="_blank">Invitrogen</a>) 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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Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of <i>alr</i> and purification using gel extraction clean-up kit from Promega (link) | Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of <i>alr</i> and purification using gel extraction clean-up kit from Promega (link) | ||
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</ul> | </ul> | ||
<ul> | <ul> | ||
| - | Transformation of the oligonucleotide containing the flanking sites for the deletion of <i>alr</i>. The successful replacement of the constitutive alanine racemase was verified via | + | <li> |
| + | Transformation of the oligonucleotide containing the flanking sites for the deletion of <i>alr</i>. The successful replacement of the constitutive 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#alr_Ec_control1" target="_blank">alr_Ec_control1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#alr_Ec_control2" target="_blank">alr_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#alr_Ec_control1" target="_blank">alr_Ec_control1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#alr_Ec_control2" target="_blank">alr_Ec_control2</a>) | ||
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| - | + | Resulting in the genotype KRX <i>kan:alr</i>, DH5aplha <i>kan:alr</i> repsectivly. | |
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</ul> | </ul> | ||
</div> | </div> | ||
Revision as of 16:13, 28 August 2014
 |
June |
 |
- Design of the Primer for the deletion of the whole coding sequence of the constitutive alanine racemase (alr) and the catabolic alanine racemase (dadX) from E. coli using the Genebridge Red/ET-System.
- Design of the Primer for the integration of the konstitutive alanine racemase alr into the pSB1C3-Backbone.
- Transformation of the E. coli strains KRX (Promega) and DH5alpha (Invitrogen) 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 alr and purification using gel extraction clean-up kit from Promega (link)
-
Transformation of the oligonucleotide containing the flanking sites for the deletion of alr. The successful replacement of the constitutive alanine racemase was verified via kanamycin selection and
Colony PCR (alr_Ec_control1, alr_Ec_control2)
- Annealing temperature: 55 °C
- Bands as expected (10 bp)
