User:J. M. Uriel Urquiza/Notebook/pRK404 & pK18Mobsacb standardization/2011/03/15

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Entry title This document describes the standardization of pK18mobsacB & pRK404 for iGEM proposes. Destroying EcoRI Origin site The linearized plasmid pRK404 was purified from an 1% agarose gel using the High Pure for PCR Kit from Roche. Then DNA concentration was quantified with Nanodrop. The lecture was 2.6 ng/µl of DNA. I concentrated my dna sample and then it was resuspended in 10µL deionized water and incubated at 65ºC for 10 min and 5 in ice. To the last sample, 3µl of T4 ligase buffer was added, 2µL of T4 DNA ligase and 5 µL of deionized water. I left the latter reaction at 9ºC for overnight incubation. Tomorrow I am going to transform DH5a and plate them in LB agar supplemented with X-Gal, and finally select thos colonies that look blue.