Restriction Digest Laboratory

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Presentation transcript:

Restriction Digest Laboratory

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.

Restriction Enzyme Digest

DNA Separation following Digest

Markers: Size Identification

RFLP provides a map of your plasmid A map gives the number and position of cutting sites A maps gives the size of fragments

Remember Plasmid is Circular Circular DNA: the number of fragments=number (N) of cutting sites versus Linear DNA: number of fragments=N+1

Linear DNA Plasmid DNA 2 cutting sites 2 fragments 2 cutting sites 3 fragments

You must carefully follow page 3-65 7 groups for today’s experiment Each group should set up a rack with the tubes necessary for the restriction digest Assign a member of your group to pick up sample tubes.

Place tubes … On ICE

Pipette the samples as shown on page 3-65

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)).

Gel Electrophoresis Load 20 ul per well Run gel at 75 volts for 45 minutes Take photograph