SEED/2008/Day 2

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
  • DNA / restriction enzymes
  • iGEM eau d'e colidemo , UT Photography
• 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