Biology > Lab Report > PCR Cloning and Expression of a GFP fusion protein in E. coli Hunter College, CUNYBIO 203Genetics La (All)

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PCR Cloning and Expression of a GFP fusion protein in E. coli Hunter College, CUNYBIO 203Genetics Lab Report

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Lab Report II (Biol 203, Spring 2017) PCR Cloning and Expression of a GFP fusion protein in E. coli PART I (No lab data required)- 60 points 1. In this lab, you have used the enzyme called T4 DNA l... igase in order to ligate a PCR-generated insert to a vector called pSK+. Regarding T4 DNA ligase: a. What is the unit definition? One unit is defined as the amount of enzyme required to provide 50% ligation of HindIII fragments of λ DNA (5’ DNA termini concentration of 0.12 m, 300 g/ml) μ μ in a total reaction volume of 20 l over a μ duration of 30 minutes incubation at a temperature of 16°C in 1x T4 DNA ligase reaction buffer. b. What is the composition of the reaction buffer? The composition of the reaction buffer is 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5 at 25°C c. How do you inactivate the enzyme? The enzyme can be inactivated by introducing heat, specifically at a temperature of 65°C for 10 minutes. 2. Here is a simple depiction of the pSK+ vector: The vector allows for protein expression in E. coli. Briefly explain the role of the following elements in the context of gene expression and protein production: LacP: LacP is the Lac Operon promoter, a regulatory sequence which contains the -10 and -35 consensus sequences that binds RNA polymerase. The binding of RNA polymerase to the LacP region is necessary for the start of transcription. [Show More]

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