Your Federal Quarterly Tax Payments are due April 15th Get Help Now >>

Restriction Fragment Length Polymorphism Lab - by ert554898


									Restriction Digest Laboratory

  Restriction fragment length polymorphism

• You 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
         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.
                         What is:
• A restriction enzyme(s)?

   – An endonuclease
   – We will focus on type II.

• A restriction digest?
Restriction Enzyme Digest
                     Examples of Restriction Enzymes

                          Links to restriction enzymes:
Gel Electrophoresis Following Digest
          Analysis of Data

Allows you to identify sizes of plasmid

By comparing migration of digested plasmid

Example of known sizes of DNA
   DNA Ladder or Markers
• A map gives the size of fragments
• A map gives the number and position of cutting sites
                                         800        600

     Not your map!                1500


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

• versus

• Linear DNA: number of fragments=N+1
Plasmid DNA         Linear DNA

  2 cutting sites      2 cutting sites
  2 fragments          3 fragments
Today’s experiment

Restriction of Digest of plasmid DNA
using two restriction enzymes.
   Please refer to page 10 of the handout
                 (6 groups)
• Each Group set up a rack with:

   –   Reaction buffer
   –   water
   –   Plasmid DNA
                              Must keep on ice
   –   Ava I
   –   SacII
   –   Loading Dye
   –   Standard (marker or ladder) DNA

• Label four microfuge tubes 1→4
Pipette the samples as shown on page in
       handout—not lab manual.
 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
                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
         Gel Electrophoresis
• Load 25 ul per well

• Run gel at 75 volts until the dye front is
  approximately half-way down gel.

• Take photograph

To top