Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602010"
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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> | + | electrophoresis, digested with <i>Dpn</i>I for 120 minutes and subsequently |
− | purified. The <I>xylC</I> amplicon was cut with <i> | + | 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
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.