Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602058"
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The <a href=" http://parts.igem.org/Part:BBa_K1602000" title="Opens internal link in current window" class="internal link">BBa_K1602000</a> part and the <a href=" http://parts.igem.org/Part:BBa_K1602001" title="Opens internal link in current window" class="internal link">BBa_K1602001</a> part were combined.</p> | The <a href=" http://parts.igem.org/Part:BBa_K1602000" title="Opens internal link in current window" class="internal link">BBa_K1602000</a> part and the <a href=" http://parts.igem.org/Part:BBa_K1602001" title="Opens internal link in current window" class="internal link">BBa_K1602001</a> part were combined.</p> | ||
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<p>Firstly the B0034-<em>xylB</em> composite was digested with <em>EcoR</em>I and <em>Spe</em>I while the B0034-<em>xylC</em> composite was digested with <em>Xba</em>I and <em>Pst</em>I. They both were taken from a pSB1A2 plasmid. The restriction products were dephosphorylated and ligated in one step into a pSB1C3 vector, which was cut with <em>EcoR</em>I and <em>Pst</em>I. <em>E. coli</em> Top 10 cells were transformed with the ligation product. The resulting colonies were screened for positive clones by restriction digest with <em>EcoR</em>I and <em>Pst</em>I. Plasmid DNA was isolated from positive clones and verified by sanger sequencing.</p> | <p>Firstly the B0034-<em>xylB</em> composite was digested with <em>EcoR</em>I and <em>Spe</em>I while the B0034-<em>xylC</em> composite was digested with <em>Xba</em>I and <em>Pst</em>I. They both were taken from a pSB1A2 plasmid. The restriction products were dephosphorylated and ligated in one step into a pSB1C3 vector, which was cut with <em>EcoR</em>I and <em>Pst</em>I. <em>E. coli</em> Top 10 cells were transformed with the ligation product. The resulting colonies were screened for positive clones by restriction digest with <em>EcoR</em>I and <em>Pst</em>I. Plasmid DNA was isolated from positive clones and verified by sanger sequencing.</p> | ||
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| + | The <a href=" http://parts.igem.org/Part:BBa_K1602011" title="Opens internal link in current window" class="internal link">BBa_K1602011</a> part and the <a href=" http://parts.igem.org/Part:BBa_K1602056" title="Opens internal link in current window" class="internal link">BBa_K1602056</a> part were combined | ||
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| + | The B0034-yagE-pSB1A2 composite was digested with SpeI and PstI while the B0034-yjhG-B0034-yqhD-pSB1A2 composite was digested with XbaI and PstI. The restriction products were ligated to a three gene composite and the resulting product was transformed into E. coli Top10. Colonies were then screened through restriction digest with EcoRI and PstI and plasmid DNA was isolated from positive clones. The resulting B0034-yagE-B0034-yjhG-B0034-YqhD-pSB1A2 composite was restricted with EcoRI and PstI and ligated into a RFP-pSB1C3 vector. The ligation product was transformed into E. coli Top 10 which were screened by CPCR using YqhD-Prefix(FW) and VR oligonucleotides. Plasmid DNA from positive clones was isolated and verified by sanger sequencing. | ||
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Revision as of 17:53, 18 September 2015
K1602016 - B0034-D-Xylose Dehydratase Plus B0034-D-Xylonolacton Lactonase (B0034-xylB-B0034-xylC)
Figure 1 Restriction digest of B0034-xylB-B0034-xylC (along with others). You can see the xylB-xylC string (below 2 kbp) and the pSB1C3 backbone (above 2 kbp). DNA marker: 2-Log DNA Ladder (NEB)..
The BBa_K1602000 part and the BBa_K1602001 part were combined.
Firstly the B0034-xylB composite was digested with EcoRI and SpeI while the B0034-xylC composite was digested with XbaI and PstI. They both were taken from a pSB1A2 plasmid. The restriction products were dephosphorylated and ligated in one step into a pSB1C3 vector, which was cut with EcoRI and PstI. E. coli Top 10 cells were transformed with the ligation product. The resulting colonies were screened for positive clones by restriction digest with EcoRI and PstI. Plasmid DNA was isolated from positive clones and verified by sanger sequencing.
BBa_K1602011 part and the BBa_K1602056 part were combined The B0034-yagE-pSB1A2 composite was digested with SpeI and PstI while the B0034-yjhG-B0034-yqhD-pSB1A2 composite was digested with XbaI and PstI. The restriction products were ligated to a three gene composite and the resulting product was transformed into E. coli Top10. Colonies were then screened through restriction digest with EcoRI and PstI and plasmid DNA was isolated from positive clones. The resulting B0034-yagE-B0034-yjhG-B0034-YqhD-pSB1A2 composite was restricted with EcoRI and PstI and ligated into a RFP-pSB1C3 vector. The ligation product was transformed into E. coli Top 10 which were screened by CPCR using YqhD-Prefix(FW) and VR oligonucleotides. Plasmid DNA from positive clones was isolated and verified by sanger sequencing.