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Restriction Digest Laboratory

Restriction Digest Laboratory. Restriction fragment length polymorphism. Reminder. You have transformed bacteria with plasmid DNA You have isolated plasmid DNA Today you will perform an RFLP analysis & Confirm your Plasmid Isolation.

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Restriction Digest Laboratory

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  1. Restriction Digest Laboratory Restriction fragment length polymorphism

  2. Reminder • You havetransformed bacteria with plasmid DNA • You haveisolated plasmid DNA • Today you will perform an RFLP analysis • & Confirm your Plasmid Isolation

  3. This is the third and final section of your lab report. • Digest plasmid DNA • Determine number of cutting sites • Determine location of cutting sites • Determine size of fragments • Present the “map” of the plasmid in your report The steps in BLUE you will complete outside of class as part of your data analysis.

  4. What is: • A restriction enzyme(s)? • An endonuclease • We will focus on type II. • A restriction digest?

  5. Restriction Enzyme Digest

  6. Examples of Restriction Enzymes Links to restriction enzymes: http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp http://www.accessexcellence.org/AE/AEC/CC/re_chart.php http://www.neb.com/nebecomm/EnzymeFinder.asp?

  7. Gel Electrophoresis Following Digest

  8. Analysis of Data Allows you to identify sizes of plasmid By comparing migration of digested plasmid To KNOWN SIZES of DNA.

  9. Example of known sizes of DNA DNA Ladder or Markers

  10. A map gives the size of fragments • A map gives the number and position of cutting sites 60 800 600 JUST AN EXAMPLE Not your map! 1500 Plasmid map 1400

  11. Remember Plasmid is Circular • Circular DNA: the number of fragments=number (N) of cutting sites • versus • Linear DNA: number of fragments=N+1

  12. Linear DNA Plasmid DNA 2 cutting sites 2 fragments 2 cutting sites 3 fragments

  13. Today’s experiment Restriction of Digest of plasmid DNA using two restriction enzymes.

  14. Please refer to page 10 of the handout(6 groups) • Each Group set up a rack with: • Reaction buffer • water • Plasmid DNA • NotI for AM lab • SfiI for AM lab • Or • AluI for PM lab • HphI for PM lab • Loading Dye • Standard (marker or ladder) DNA • Label four microfuge tubes 1→4 Must keep on ice

  15. Pipette the samples as shown on page in handout—not lab manual.

  16. After you are finished pipetting your samples • Place samples at 37C for 1 hour • After 1 hour you will be ready to load your gel

  17. Restriction Digest • AFTER 1 hour DIGESTION: You must add 5 ul 10X loading dye to your samples (not to the ladder (L)). • Pre-heat all samples including ladder for 3-5 min. at 65C

  18. Gel Electrophoresis • Load 25 ul per well • Run gel at 75 volts until the dye front is approximately half-way down gel. • Take photograph

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