Download - Restriction Digest Laboratory
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Restriction Digest Laboratory
Restriction fragment length polymorphism
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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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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.
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What is:• A restriction enzyme(s)?
– An endonuclease– We will focus on type II.
• A restriction digest?
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Restriction Enzyme Digest
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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?
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Gel Electrophoresis Following Digest
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Analysis of Data
Allows you to identify sizes of plasmid
By comparing migration of digested plasmid
To KNOWN SIZES of DNA.
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Example of known sizes of DNA DNA Ladder or Markers
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• A map gives the size of fragments
• A map gives the number and position of cutting sites
JUST AN EXAMPLENot your map!
Plasmid map
1500
80060
600
1400
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Remember Plasmid is Circular
• Circular DNA: the number of fragments=number (N) of cutting sites
• versus
• Linear DNA: number of fragments=N+1
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2 cutting sites2 fragments
2 cutting sites3 fragments
Plasmid DNA Linear DNA
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Today’s experiment
Restriction of Digest of plasmid DNAusing two restriction enzymes.
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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
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Pipette the samples as shown on page in handout—not lab manual.
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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
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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
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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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