restriction digest laboratory restriction fragment length polymorphism

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  • Restriction Digest Laboratory

    Restriction fragment length polymorphism

  • ReminderYou have transformed bacteria with plasmid DNA

    You have isolated plasmid DNA

    Today you will perform an RFLP analysis& Confirm your Plasmid Isolation

  • This is the third and final section for RESULTS that will be part of your in-lab report interpretation.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 reportThe steps in BLUE you will complete outside of class as part of your data analysis.

  • What is:A restriction enzyme(s)?

    An endonucleaseWe will focus on type II.

    A restriction digest?

  • Restriction Enzyme Digest

  • Examples of Restriction Enzymes

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

  • Gel Electrophoresis Following Digest

  • Analysis of DataAllows you to identify sizes of plasmid

    By comparing migration of digested plasmid

    To KNOWN SIZES of DNA.

  • Example of known sizes of DNA DNA Ladder or Markers

  • A map gives the size of fragmentsA map gives the number and position of cutting sitesJUST AN EXAMPLENot your map!Plasmid map1500 800606001400

  • Remember Plasmid is CircularCircular DNA: the number of fragments=number (N) of cutting sites

    versus

    Linear DNA: number of fragments=N+1

  • 2 cutting sites2 fragments2 cutting sites3 fragmentsPlasmid DNALinear DNA

  • Todays experimentRestriction of Digest of plasmid DNAusing two restriction enzymes.

  • Please refer to page 10 of the handout(6 groups)Each Group set up a rack with:

    Reaction bufferwaterPlasmid DNANotI for AM labSfiI for AM lab

    Or

    AluI for PM labHphI for PM labLoading Dye

    Standard (marker or ladder) DNA

    Label four microfuge tubes 14Must keep on ice

  • Pipette the samples as shown on page in handoutnot lab manual.

  • After you are finished pipetting your samplesPlace samples at 37C for 1 hour

    After 1 hour you will be ready to load your gel

  • Restriction DigestAFTER 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

  • Gel ElectrophoresisLoad 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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