Lab 6 Restriction mapping

Lab 6 Restriction mapping - Q1 (2pts): Why is it necessary...

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Unformatted text preview: Q1 (2pts): Why is it necessary to cut a plasmid with a restriction enzyme before its size can be determined by agarose gel electrophoresis? Q2 (3pts): What information about a plasmid do we need in order to confirm its identity accurately by restriction mapping? Why? Lab #6 Plasmid Identity by Restriction Mapping Objectives: to run agarose gel electrophoresis of restriction enzyme digested pGEM and pGEM- luc to analysis restriction digested fragments pellet cells pGEM pGEM- luc restriction digest Plasmid isolation Approaches to Confirm Plasmid Identity 1. Restriction enzyme digestion followed by gel electrophoresis of digested fragments 2. DNA sequencing Last Week: 3 major forms of plasmid DNA C. Supercoiled: compact shape, no excessive damage (fastest) A. Relaxed or Nicked Circular: nicks (on one strand of DNA) in the supercoiled plasmid (slowest) B. Linear : direct opposite strand nicks or cuts by restriction enzyme C. Highly supercoiled: has more superhelical turns The size of DNA is determined by looking at its linear form and compare it to a DNA marker. Predicting restriction digest patterns from a...
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Lab 6 Restriction mapping - Q1 (2pts): Why is it necessary...

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