Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602013"
Line 8: | Line 8: | ||
<figcaption><br><b>Figure 1</b> PCR of cadA (X). The size of the amplified product was around 1.5 kbp. DNA marker: 2-Log DNA Ladder (NEB).</figcaption> | <figcaption><br><b>Figure 1</b> PCR of cadA (X). The size of the amplified product was around 1.5 kbp. DNA marker: 2-Log DNA Ladder (NEB).</figcaption> | ||
</figure> | </figure> | ||
− | The <i>YqhD</i>-gene was isolated from K12 E.coli and amplified through colony PCR using the <i>YqhD</i> (FW) and <i>YqhD</i> (REV) oligonucleotides. The PCR product was verified by agarose gel electrophoresis, digested with | + | The <i>YqhD</i>-gene was isolated from K12 E.coli and amplified through colony PCR using the <i>YqhD</i> (FW) and <i>YqhD</i> (REV) oligonucleotides. The PCR product was verified by agarose gel electrophoresis, digested with <i>Dpn</i>I for 120 minutes and subsequently purified. The <i>YqhD</i> amplicon was cut with <i>EcoR</i>I and <i>Pst</i>I and ligated into a pSB1C3 vector using T4-ligase.<i>E.Coli</i> Top10 has been transformed with the ligation product by heat shock transformation and the cells were spread out on a CMP agar plate. Clones were screened with colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones which were verified by sanger sequencing. |
</div></html> | </div></html> |
Revision as of 16:40, 17 September 2015
K1602013 - aldehyde reductase YqhD
![](https://static.igem.org/mediawiki/2015/8/86/Agar_gel_c3_ cadA.png)
Figure 1 PCR of cadA (X). The size of the amplified product was around 1.5 kbp. DNA marker: 2-Log DNA Ladder (NEB).