"
Team:Bielefeld-CeBiTec/Notebook/Journal/Biosafety/Jun
From 2014.igem.org
(Difference between revisions)
| Line 50: | Line 50: | ||
<div class="content"> | <div class="content"> | ||
| - | < | + | <ul>Design of the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Deletion_alanine_racemase" target="_blank">Primer</a> for the deletion of the whole coding sequence of the constitutive alanine racemase (<i>alr</i>) and the catabolic alanine racemase (<i>dadX</i>) from <i>E. coli</i> using the <a href="http://www.genebridges.com/storage/Manuals_PDF/K006%20Ecoli%20Gene%20Deletion%20Kit-version2.3-2012.pdf" target="_blank">Genebridge Red/ET-System</a>. |
| - | </ | + | </ul> |
</div> | </div> | ||
| Line 69: | Line 69: | ||
</div> | </div> | ||
<div class="content"> | <div class="content"> | ||
| - | < | + | <ul> |
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. | ||
| - | </ | + | </ul> |
</div> | </div> | ||
</div> | </div> | ||
| Line 88: | Line 88: | ||
</div> | </div> | ||
<div class="content"> | <div class="content"> | ||
| - | < | + | <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> |
</div> | </div> | ||
</div> | </div> | ||
| Line 106: | Line 106: | ||
</div> | </div> | ||
<div class="content"> | <div class="content"> | ||
| - | < | + | <ul> |
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) | ||
| - | </ | + | </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 | 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 | ||
| Line 121: | Line 121: | ||
and kanamycin selection. Resulting in the genotype KRX <i>kan:alr</i>, DH5aplha <i>kan:alr</i> repsectivly. | and kanamycin selection. Resulting in the genotype KRX <i>kan:alr</i>, DH5aplha <i>kan:alr</i> repsectivly. | ||
| - | </ | + | </ul> |
</div> | </div> | ||
</div> | </div> | ||
Revision as of 16:09, 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
Colony PCR (alr_Ec_control1, alr_Ec_control2)
- Annealing temperature: 55 °C
- Bands as expected (10 bp)
