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

Restriction Fragment Length Polymorphism Lab - smc.edu by ert554898

VIEWS: 6 PAGES: 20

									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
 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:


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?
Gel Electrophoresis Following Digest
          Analysis of Data

Allows 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 fragments
• A map gives the number and position of cutting sites
                                               60
                                         800        600




     JUST AN EXAMPLE
     Not your map!                1500



                                                          1400

        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