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BIOB12H3 (30)
Lecture 5

Laboratory Exercise - Week 5.pdf

6 Pages
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Department
Biological Sciences
Course Code
BIOB12H3
Professor
Daman Bawa

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©Brunt 2011
1
Lab 5A: Restriction Enzyme Digest of plasmid DNA isolated in Lab 4B
In today’s lab, you will set up a restriction enzyme digest on an aliquot of your
plasmid DNA for Lab 4B and separate the product of digestion by agarose gel
electrophoresis.
Restriction enzymes are found in nature in prokaryotes . Molecular Biologists
have cloned the genes encoding these restriction endonucleases and these enzymes
are now readily available from commercial sources. Hundreds of restriction enzymes
can be purchased from commercial sources. The type II restriction endonucleases
recognize short, palindromic sequences and make two single stranded cuts at the target
site. These may generate 5’ overhangs, 3’ overhangs or blunt ends. Examples of each
of these are shown below.
Eco RI (5’ overhang) Pst I (3’ overhang) Sca I (blunt ends)
5’-GAATTC-3’ 5’-CTGCAG-3’ 5’-AGTACT-3’
3’-CTTAAG-5’ 3’-GACGTC-5’ 3’-TCATGA-5’
5’-G 5’-CTGCA-3’ 5’-AGT
3’-CTTAAG-5’ 3’-G 3’-TCA
Restriction enzymes are commonly employed for DNA gel blots (Southern blots) to
reproducibly fragment large chromosomal DNA molecules, and these enzymes are
important in molecular cloning to prepare both the vector and the target DNA molecules
for DNA ligation. Often after the subcloning of a fragment from a larger DNA molecule,
restriction enzymes are used to determine if the proper fragment has been cloned. In
some cases, large DNA fragments are subjected to restriction enzyme mapping to
determine the linear order of restriction sites ( you will do this in the next lab period).
This is useful in determining the orientation of the cloned fragment in the vector and the
information can be used to decide on the subcloning strategy (i.e., which enzymes
might be useful for generating small pieces for subcloning).
Electrophoresis
For most purposes, agarose gel electrophoresis is the method used to separate DNA
fragments. Agarose is a neutral polysaccharide isolated from seaweed, which when
cooled forms a solid matrix that will carry an electrical current. DNA molecules have a
phosphate backbone and thus carry a net negative charge. During electrophoresis
they move towards the positive electrode. When a gel is cast or poured, the agarose is
©Brunt 2011
2
dissolved in the appropriate buffer [usually 0.5X TBE (Tris, boric acid, EDTA)] by
heating (in a microwave oven or on a hot plate), and after it cools to approximately 50-
60°C, ethidium bromide or “safe ethidium bromide” is added and the gel is poured into a
mold. A comb is inserted which creates ‘wells’. When the comb is removed, these wells
are observed as small rectangular troughs.
Small DNA fragments can easily move through the gel, but larger molecules
encounter more resistance and therefore migrate more slowly. In most cases, one also
runs a molecular size standard on the gel (we will use the 1kb ladder which is
commercially purchased). The migration of these standard fragments through the
gel can be measured and a graph is constructed which plots the distance
migrated versus the logarithm of the molecular weight. By measuring the distance
migrated of the unknown and correlating this to the plot of the standards, one
can determine the molecular weight of the unknown..
After you run your gel (which contains Ethidium Bromide) , you can visualize the DNA
fragments by placing your gel on a UV light box. Incorporated into the gel is the dye
ethidium bromide. The ethidium bromide is a small molecule that intercalates between
the DNA bases and upon excitation with ultraviolet (UV) light, it fluoresces orange. This
gives the DNA fragments an orange appearance against a black background. When a
photograph is taken with a digital camera and the appropriate filter the DNA bands
appear white against a black background.
Ethidium bromide is a carcinogen . Always wear gloves and dispose of the gel
in the appropriate location for ethidium bromide waste. There are newer
products that have recently come onto the marker that are reported to have the
same sensitivity and are not carcinogenic .
UV light can damage your skin and eyes . You must wear a UV face shield
that is rated to prevent UV penetration

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Description
Brunt 2011Lab 5A Restriction Enzyme Digest of plasmid DNA isolated in Lab 4BIn todays lab you willset up a restriction enzyme digeston analiquot of your plasmid DNA for Lab 4Band separate the product of digestion by agarose gel electrophoresisRestriction enzymes are found in nature in prokaryotes Molecular Biologists have cloned the genes encoding these restriction endonucleases and these enzymes are now readily available from commercial sources Hundreds of restriction enzymes can be purchased from commercial sourcesThe type II restriction endonucleases recognize short palindromic sequences and make two single stranded cuts at the target siteThese may generate 5 overhangs 3 overhangs or blunt endsExamples of each of these are shown belowEco RI 5 overhangPst I 3 overhangSca I blunt ends 5GAATTC35CTGCAG35AGTACT3 3CTTAAG53GACGTC53TCATGA55G 5CTGCA35AGT3CTTAAG53G 3TCA Restriction enzymes are commonly employed for DNA gel blots Southern blots to reproducibly fragment large chromosomal DNA molecules and these enzymes are important in molecular cloningto prepare both the vector and the target DNA molecules for DNA ligation Often after the subcloning of a fragment from a larger DNA molecule restriction enzymes are used to determine if the proper fragment has been clonedIn some cases large DNA fragments are subjected to restriction enzyme mapping to determine the linear order of restriction sitesyou will do this in the next lab periodThis is useful in determining the orientation of the cloned fragment in the vector and the information can be used to decide on the subcloning strategy ie which enzymes might be useful for generating small pieces for subcloning ElectrophoresisFor most purposes agarose gel electrophoresis is the method used to separate DNA fragmentsAgarose is a neutral polysaccharide isolated fromseaweed which when cooled forms a solid matrix that will carry an electrical currentDNA molecules have a phosphate backbone and thus carry anet negative chargeDuring electrophoresis they move towards the positive electrode When a gel is cast or poured the agarose is 1
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