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
JUST AN EXAMPLE
Not your map!
600
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
2 cutting sites
2 fragments
Linear DNA
2 cutting sites
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:
–
–
–
–
–
–
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Reaction buffer
water
Plasmid DNA
Must keep on ice
PVU 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