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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Restriction Digest Laboratory

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Restriction digest laboratory

Restriction Digest Laboratory


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.


Restriction enzyme digest

Restriction Enzyme Digest


Dna separation following digest

DNA Separation following Digest


Markers size identification

Markers: Size Identification


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


Restriction digest laboratory

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


Place tubes

Place tubes …

  • On ICE


Pipette the samples as shown on page 3 65

Pipette the samples as shown on page 3-65


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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