Restriction digest laboratory
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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

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Presentation Transcript

Reminder
Reminder

  • You havetransformed bacteria with plasmid DNA

  • You haveisolated plasmid DNA

  • Today you will perform an RFLP analysis

  • & Confirm your Plasmid Isolation


This is the third and final section of your lab report
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.





Rflp provides a map of your plasmid
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
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
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
Obtain a rack and:

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


2 place these tubes also on your rack
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




After you are finished pipetting your samples
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
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
Gel Electrophoresis

  • Load 25 ul per well

  • Run gel at 75 volts for 45 minutes

  • Take photograph


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