25 Jul WHAT DO WHITE COLONIES SIGNIFY?
You are cloning human antithrombin in the lab using a plasmid pBlueScript that contains an ampicil Show more You are cloning human antithrombin in the lab using a plasmid pBlueScript that contains an ampicillin resistance marker (ampR) and a lacZ gene interrupted by a multiple cloning site. You transformed the ligation products into competent E. coli cells by electroporation then plated electroporated E. coli on agar plates containing ampicillin and X-Gal and waited one day until colonies are visible. You use blue/white screening to help you identify clones that carry antithrombin. You observe numerous blue and white colonies on the agar plate. 1) What do white colonies signify? Briefly explain your answer. 2) What do blue colonies signify? Briefly explain your answer. 3) Which colonies (blue white or both) would you want to pick for further analysis to check for the successful cloning of the antithrombin gene? Briefly explain your answer. 4) If you forgot to add X-gal to the agar selection medium how would the colonies that grow differ phenotypically from the ones that grow in plates with X-Gal? Briefly explain your answer. 5) If you forgot to add ampicillin to the agar medium what other colonies would grow that wont normally grow in plates with ampicillin? What color would those other colonies most likely be? Briefly explain your answer. 6) Research and describe the role of the synthetic compound IPTG in blue-white screening. What naturally occurring compound does it mimic or replace? Show less
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