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Lecture 5

Laboratory Exercise - Week 5.pdf

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Biological Sciences
Daman Bawa

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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