Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602058"
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Revision as of 18:43, 18 September 2015
K1602058 - B0034-2-Dehydro-3-Deoxy-D-Pentonate Aldolases-B0034-D-Xylonic Acid Dehydratase and B0034-Aldehyde Reductase (B0034-yagE-B0034-yjhG-B0034-yqhD)
Figure 1 Restriction digest of B0034-yagE-B0034-yjhG-B0034-YqhD (along with others). You can see the yagE-yjhG-YqhD string (above 3 kbp) and the pSB1C3 backbone (above 2 kbp). DNA marker: 2-Log DNA Ladder (NEB).
.The 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.