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Restriction Digest

Today: Setup digest and incubate


Before Thanksgiving: we will run
the gel electrophoresis and
analyze the results

What are restriction


enzymes?

Enzymes that are


able to cleave at
specific and unique
DNA sequences

Produced by bacteria
as a defense against
bacteriophage and
other foreign DNA

Named according to
the bacteria that
produce them

Restriction sites

Restriction enzymes (RE) recognize specific


sequences called restriction sites (usually palindromic
sequences e.g. TTCATGAA)
The longer the restriction site sequence the enzyme
recognizes the less likely the sequence will occur more
than once
Two types of ends are produced when a RE cuts DNA:
1. Blunt
2. Sticky

Enzymes used in our lab


EcoRI (from Escherichia coli strain R)
5-NNNGAATTCNNN-3
3-NNNCTTAAGNNN-5

5-NNNG-3
3-NNNCTTAAG-5

5-AATTCNNN-3
3-GNNN-5
Sticky Ends

BamHI (from Bacillus amyloliquefaciens)


5-NNNGGATCCNNN-3
3-NNNCCTAGGNNN-5

5-NNNG-3
5-GATTCNNN-3
3-NNNCCTAG-5
3-GNNN-5
Sticky Ends

HindIII (from Haemophilus influenzae)


5-NNNAAGCTTNNN-3
3-NNNTTCGAANNN-5

5-NNNA-3
3-NNNTTCGA-5

http://www.phschool.com/science/biology_place/biocoach/red/cleave1.html
http://highered.mheducation.com/olc/dl/120078/bio37.swf

5-AGCTTNNN-3
3-ANNN-5
Sticky Ends

What are plasmids?

Small, self-replicating (has


origin of replication), extrachromosomal, circular,
double stranded DNA
3,000 8,000 bp long
Often found in bacteria
plasmids carrying genes
for antibiotic resistance
pAMP plasmid carries a
gene for resistance to
ampicilin

Uses of
plasmids

Genetic
Engineering,
Biotechnology
or more
specifically
Gene cloning

Our experiment

Overall Purpose: to execute a restriction digest,


analyze the results from a gel electrophoresis, in
order to construct a map of pAMP plasmid

Experimental design:
1. Setup the digestion of a plasmid using three
restriction enzymes: EcoRI, BamHI and HindIII
2. Run samples on an agarose gel electrophoresis
3. Perform analysis of the results and construct a
restriction map

Digestion of plasmid DNA with


EcoRI, BamHI and HindIII

Tube # 1 DNA control - no enzyme added


Tube # 2 DNA Digested with BamHI only
Tube # 3 DNA Digested with BamHI + EcoRI
Tube # 4 DNA Digested with BamHI + HindIII
Tube # 5 DNA Digested with HindIII + EcoRI
Tubes were incubated at 37C degrees for one hour and
stored at 4C degrees.

Gel Electrophoresis

Gel-like matrix (agarose or acrylamide) is used to separate


molecules based on their size, with the use of an electric current

DNA is negatively charged so if electric current is applied DNA will


migrate towards the positive pole

Acrylamide+Bisacrylamide gels are mostly used to separate proteins and


nucleic acids
Agarose gels are used to separate nucleic acids

Thus, it is important to set up your electrodes correctly when running a gel.

Shorter DNA fragments migrate faster across the gel than longer
DNA fragments which migrate slower
To determine the actual size of the fragments we use known
standards (called marker or ladder)

Agarose Gel Electrophoresis:


Making the Gel
add Gelstar DNA stain, then

Gel tray will be perpendicular, once the gel has solidified, place the
gel tray parallel with the gel box, pour the running buffer in the gel box
to cover the gel, then slowly remove the comb. Add you samples to
the wells, making note of which well each sample is in.

Setting up the Apparatus

DNA is negatively
charged so if
electric current is
applied DNA will
migrate towards
the positive pole
Thus, it is
important to set up
your electrodes
correctly when
running a gel
Red is positive
Black is negative

Loading DNA into the Gel


Before we load the
samples on the gel we
need to add tracking dye.
The tracking dye is added
to make the solution dense
so that our sample will
"sink" into the well and to
make the samples visible
during analysis
Load samples into the
wells using a micropipettor

Movement of DNA Fragments


We will run the gel at 90V
for about one hour, until the
tracking dye migrates
about 2/3 of the way
through the gel
At this stage the DNA
band(s) cannot be seen,
only the tracking dye is
visible

Photographing the Results


In order to see the bands of
DNA we have stained the gel
with a fluorescent dye which
binds to DNA (Gelstar DNA
stain) making the bands
visible under UV light
We will use the UVP
illuminator to take a
photograph of the gel

Analysis of the results and plasmid


map construction
1. Determine the
approximate
sizes of the
fragments
generated in
each tube
2. Construction of
the restriction
map of the
plasmid

Predicted
restriction sites

3000 bp
2500 bp

500 bp

http://www.wiley.com/legacy/college/boyer/0470003790/animations/agarose/agarose.htm
http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html

Plasmid X
3000 bp