Restriction Digest Laboratory

Restriction fragment length polymorphism

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 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.

What is:

• A restriction enzyme(s)?

– An endonuclease– We will focus on type II.

• A restriction digest?

Restriction Enzyme Digest

 

Examples of Restriction Enzymes

http://www.accessexcellence.org/AE/AEC/CC/re_chart.php

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp

Links to restriction enzymes:

http://www.neb.com/nebecomm/EnzymeFinder.asp?

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!

Plasmid map

1500

80060

600

1400

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

Today’s experiment

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

– Or

– 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

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 until the dye front is approximately half-way down gel.

• Take photograph