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Team:TU Darmstadt/Notebook/Labjournal/K1497003
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Revision as of 14:54, 17 October 2014
K1497003 - Chalcone Isomerase with strong RBS (B0034-CHI)
After creating Bba_K1497000, we added a strong RBS (Bba_B0034) in front of the coding part of CHI. For this reason we used PCR with primers coding for RBS, Biobrick prefix and Biobrick suffix (RBS_CHI_pre and CHI_psb1c3_Suf2). We analyzed the PCR via agarose gel electrophoresis and cleaned up positive samples with DpnI digestion and Wizard PCR Clean Up Kit (Promega). We cut the cleaned up samples with EcoRI and PstI and ligated them with a previously digestion of mRFP-pSB1C3 with EcoRI + PstI (already dephosphorylated). We transformed the ligation in Top10 E. coli via heat shock method and spread out on CMP-LB agar plates. After incubating overnight, we analyzed obtained colonies via 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.
