restriction digest laboratory restriction fragment length polymorphism
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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 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 report
The steps in BLUE you will complete outside of class as part of your data analysis.
• 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
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
JUST AN EXAMPLENot your map!
Remember Plasmid is Circular
• Circular DNA: the number of fragments=number (N) of cutting sites
• Linear DNA: number of fragments=N+1
2 cutting sites2 fragments
2 cutting sites3 fragments
Plasmid DNA Linear DNA
Restriction 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 buffer– water– Plasmid DNA– NotI for AM lab– SfiI for AM lab
– 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
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
• 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
• Load 25 ul per well
• Run gel at 75 volts until the dye front is approximately half-way down gel.
• Take photograph