Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602010"

Line 11: Line 11:
 
through PCR using the <I>xylC</I> (FW) and <I>xylC</I> (REV)
 
through PCR using the <I>xylC</I> (FW) and <I>xylC</I> (REV)
 
oligonucleotides. The PCR product was verified by agarose gel
 
oligonucleotides. The PCR product was verified by agarose gel
−
electrophoresis, digested with <i>Dpn1</i> for 120 minutes and subsequently
+
electrophoresis, digested with <i>Dpn</i>I for 120 minutes and subsequently
−
purified. The <I>xylC</I> amplicon was cut with <i>EcoRI</i> and <i>PstI</i> and
+
purified. The <I>xylC</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> Top 10
 
ligated into a pSB1C3 vector using T4-ligase. <I>E.coli</I> Top 10
 
has been transformed with the ligation product by heat shock
 
has been transformed with the ligation product by heat shock

Revision as of 16:37, 17 September 2015

K1602010 - D-xylonolactone lactonase xylC



Figure 1 PCR of cadA (X). The size of the amplified product was around 1.5 kbp. DNA marker: 2-Log DNA Ladder (NEB).
The xylC-gene was synthesized by GeneArt and amplified through PCR using the xylC (FW) and xylC (REV) oligonucleotides. The PCR product was verified by agarose gel electrophoresis, digested with DpnI for 120 minutes and subsequently purified. The xylC amplicon was cut with EcoRI and PstI and ligated into a pSB1C3 vector using T4-ligase. E.coli Top 10 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 by colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones which were verified by sanger sequencing.