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

2 cutting sites2 fragments

2 cutting sites3 fragments

Plasmid DNA Linear DNA

You must carefully follow page 3-65

• 6 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.

Obtain a rack and:

●1. Obtain new microfuge tubes and label 2-8

2. Place these tubes also on your rack

Tube L= Ladder “known sizes of DNA” Tube P=Plasmid DNA “cocktail”

Tube A: AfaI Tube B: Mae I Tube C: Xma I Tube D: Loading Dye Tube W: Water

note these enzymes are different than your lab manual

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

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 for 45 minutes

• Take photograph