Team:TU Darmstadt/Notebook/Labjournal/K1497007
From 2014.igem.org
K1497007 - Naringenin operon
For combing all for four genes of our operon, we used the previously obtained Biobricks B0034-CHS-B0034-CHI-pSB1C3 (K1497005) and B0034-4CL-B0034-TAL-pSB1C3 (K1497006). We digested B0034-4CL-B0034-TAL-pSB1C3 with EcoRI + SpeI and B0034-CHS-B0034-CHI-pSB1C3 with EcoRI + XbaI. Furthermore we dephosphorylated the digestion of B0034-CHS-B0034-CHI-pSB1C3. We ligated both digestions with T4 ligase and transformed the products into Top10 E.coli via heat shock. We spread out on CMP-LB agar plates and incubated the plates overnight at 37°C. We analyzed obtained colonies by colony PCR and agarose gel electrophoresis. Positive colonies were inoculated in 5 ml LB with CMP and plasmids were isolated after incubating for 16 h at 37°C. For further verification we send the samples to Eurofins for sequencing.