EVERGREEN INS 2008 - Lab 3 Restriction Digestion of Plasmid DNA and Gel Electrophoresis (4 pages)

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Lab 3 Restriction Digestion of Plasmid DNA and Gel Electrophoresis



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Lab 3 Restriction Digestion of Plasmid DNA and Gel Electrophoresis

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Pages:
4
School:
Evergreen State College
Course:
Ins 2008 - Forest Through Time and Space

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1 Restriction Digestion of Plasmid DNA and Gel Electrophoresis 2007 08 INS Winter Quarter Lab 3 Goals You will learn to a work with DNA b use DNA modifying enzymes and c separate DNA fragments by size using agarose gel electrophoresis To enhance this experience it is advised that you complete the prelab worksheet provided at the end of this manual Restriction enzymes are produced by bacteria to prevent foreign DNA and virus sequences from surviving in the cell These are endonucleases meaning they cut both strands of DNA at some location other than the end The counterpart to this would be an exonuclease which would digest a DNA molecule starting from an already cut end The value of many restriction enzymes is that they cut at specific base sequences and nowhere else These recognition sites are usually palindromes and can be from 4 to 15 bases in length The two enzymes we will use today are SacII Streptomyces achromogenes second II enzyme isolated and XhoI Xanthomonas holcicola first I enzyme isolated The XhoI enzyme cuts indicated by the arrow at the sequence C TCGAG while SacII cuts at the sequence CC GC GG Today you will be cutting the 5601 base pair bp plasmid pCDNA3 1Hygro with these enzymes The plasmid contains one recognition site for each enzyme The XhoI enzyme cuts at nucleotide 986 whereas the SacII cuts at nucleotide 2900 on the plasmid To determine the number and lengths of the product DNA fragments we will use agarose gel electrophoresis At normal pH values DNA molecules carry a large number of negative charges they are polyanions If the DNA molecules are placed in an electric field opposite charges attract so the DNA will move towards the positive electrode and away from the negative electrode This technique of separating molecules by their movement in an electric field is called electrophoresis Since the number of negative charges on a DNA molecule increases in proportion to its mass the acceleration on all molecules is roughly the same Large pieces



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