User:J. M. Uriel Urquiza/Notebook/pRK404 & pK18Mobsacb standardization/2011/03/15: Difference between revisions
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(Autocreate 2011/03/15 Entry for User:J._M._Uriel_Urquiza/Notebook/pRK404_&_pK18Mobsacb_standardization) |
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This document describes the standardization of pK18mobsacB & pRK404 for iGEM proposes. | 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. | |||
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Revision as of 18:37, 15 March 2011
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Entry titleThis document describes the standardization of pK18mobsacB & pRK404 for iGEM proposes. Destroying EcoRI Origin siteThe 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. | |
