SEED/2008/Day 2

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Contents

Morning

The purpose of this exercise is to learn to run a restriction digest and an agarose gel to obtain information about a DNA construct.

For each of the two DNA samples, you will set up 4 restriction digests using combinations of the enzymes EcoRI and PstI:

  • 20ul reaction:
    • 2ul NEB2
    • 0.2ul BSA
    • 2ul DNA (~500ng)
    • 16ul DI water
    • 0.5ul per enzyme
  • 4 reactions
    1. no enzyme
    2. EcoRI only
    3. PstI only
    4. EcoRI and PstI
  • Incubate at 37C over lunch

Prepare and pour a 1% agarose gel with 10-well combs.

Afternoon

  • Run a gel
    • Cover gel with buffer
    • Remove gel combs
    • Add gel loading buffer to your restriction digests
    • Carefully load samples
    • Load the DNA ladder
  • look at plates from Day 1 and take quiz
  • Image the gel and analyze bands

Lecture

  • BioBricks & Registry of Standard Biological Parts
  • Central Dogma Pardigm
    • DNA->RNA->protein (an enzyme that makes scent)
    • promoters (transcription), RBS (translation)

Instructor Preparation

  • Prepare minipreps of several different BioBrick constructs in the same plasmid but with noticeably different insert lengths
  • Grow up batch culture of registry:Part:BBa_J45200 with 10mM isoamyl alcohol
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