Biology > Lab Report > Hunter College, CUNY - BIO 203Genetics lab 1. (All)

Hunter College, CUNY - BIO 203Genetics lab 1.

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-Lab Report I (Biol 203, Spring 2019) NAME Sessions 1,2,4,5,6,7 PART I (No lab data required) 60 pts. 1. In the 203 laboratory, you have used an enzyme called T4 DNA ligase. Answer the few question... s below regarding this enzyme: a) what is the definition of one Unit for this enzyme? One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer. b) what are the optimal reaction conditions recommended by the manufacturer (buffer and incubation conditions)? Did you use these conditions in lab? The reaction conditions recommended by the manufacturer are 1X T4 DNA Ligase Reaction Buffer and Incubate at 16°C. We did use these conditions in the lab. c) can the enzyme be inactivated? If so, how? Yes it can by heating the reaction to 65 degrees Celsius. d) in the protocol recommended by the manufacturer (NEB): -what is the recommended reaction volume? 1/10 volume where the final volume should be 10X the ligase buffer. - how many units of enzyme (at 400,000 U/ml) per reaction are recommended? 20 units of T4 DNA ligase are used per 1μg of vector DNA. -what is the molar ratio of vector: insert? Which is in excess? Between 1:1 and 1:10 are recommended, 1:3 is typical. The insert will be in excess. [Show More]

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