GT CHEM 4581 - Purification and Characterization of a DNA Plasmid

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CHEM 4581 1 Purification and Characterization of a DNA Plasmid – Part B CHEM 4581: Biochemistry Laboratory I Version: January 18, 2008 INTRODUCTION Biological macromolecules that have been isolated and purified from a heterogeneous mixture must be characterized to assess the quality and effectiveness of the purification scheme and to verify the identity of the purified material. A widely used approach for characterizing purified DNA is to perform restriction mapping. Restriction mapping is similar to solving a jigsaw puzzle. The “pieces of the puzzle” are the lengths of the restriction digests. When arranged properly, the experimentally determined digests match the theoretical fragment lengths so that the identity of the DNA can be confirmed. Significant deviation from the anticipated fragment lengths would dispute the DNA identity confirmation. Digestion. Restriction endonucleases are enzymes that cut DNA at specific sites within the DNA sequence. While each restriction enzyme has a characteristic primary recognition sequence at which it preferentially cuts, most restriction enzymes are capable of cutting at secondary sites as well. Useful sources of information on restriction enzymes can be found on the web sites of commercial producers such as New England Biolabs and Invitrogen. Once DNA has been cleaved with various restriction enzymes, the sizes of the resulting fragments need to be determined. Typically fragment size determination is performed by agarose gel electrophoresis. Electrophoresis. Nucleic acids can be separated on the basis of size from ~100 base pairs to ~200,000 base pairs on agarose gels. Samples are prepared by mixing them with a tracking dye that the biochemist uses to assess the progress of the electrophoresis. Samples are then loaded onto a gel that is submerged in a buffered solution. The gel is positioned between electrodes and a power supply is attached to the gel apparatus to supply the necessary voltage for the migration. Smaller fragments of DNA will migrate farther through the gel than larger ones. A molecular weight marker is generally run for each gel for subsequent estimation of DNA fragment size. Agarose gels of nucleic acids are typically stained with Ethidium Bromide (EtBr). EtBr is a known mutagen and a potential carcinogen, therefore, care must be taken when handling it. EtBr is a fluorescent dye that intercalates into DNA between base pairs. The fluorescence of EtBr is enhanced when intercalated into DNA. EtBr binding to DNA is not sequence specific, however, the amount of fluorescence enhancement is proportional to the amount of DNA. Mapping. A “restriction map” is a depiction of how fragments of DNA from a particular digest sum. If the map of actual data matches that of the theoretical pattern, then the sequence of the DNA is verified. There are many programs available for performing theoretical restriction mapping. We will use the program NEBcutter 2.0*. To use this tool, you must minimally paste the sequence of pYP** into the designated area, select “circular” as the type of DNA, and select all NEB enzymes for use in cutting. The result will give an exhaustive list of every restriction enzyme sold by NEB that cuts the sequence provided in any place. *http://tools.neb.com/NEBcutter2/index.php **Sequence of pYP can be found as a link on the course home page.CHEM 4581 2 GOAL In the E. coli cells being used for this study, there are numerous types of DNA – genomic DNA, plasmid DNA, etc. During the previous laboratory period, a midi-prep purification of DNA (presumed to be pYP) should have resulted in 10-200 g of purified plasmid DNA. The specific aim of today’s study is to characterize the material purified from the Midi-prep purification. You want to determine how pure was the resulting DNA solution and is the purified DNA actually pYP. MATERIALS AND REAGENTS Purified DNA presumably containing pYP plasmid (from previous lab) Resriction Enzymes (each from New England Biolabs)  Ear I  EcoRI  Dra I  Hae II  10X Buffer containing: o 10 mM Tris-HCl, pH 7.9 o 50 mM NaCl o 10 mM MgCl2 o 1 mM Dithiothreitol (DTT)  Buffer for Hae II only is same as above spiked with bovine serum albumin (BSA) Agarose Gel Electrophoresis  High melting DNA agarose (note source)  MW Marker: pBR322 DNA - BstN I digest (New England Biolabs) o 1857 bp o 1058 bp o 929 bp o 383 bp o 121 bp (this band might be faint)  Primo Horizontal Submarine Gel System (Thermo Electron)  Tris-Acetate-EDTA (TAE) Buffer  Sample Loading Dye: 0.25%bromophenol blue, 0.25% xylene cyanol, 6X TAE  Power supplies  10 mg/mL ethidium bromide (1mL)  Fotophoresis UV Transilluminator (Fotodyne)CHEM 4581 3 EXPERIMENTAL PROCEDURE NOTE: You must wear gloves throughout this lab to protect your DNA. Restriction Digestion 1. Generate a table to use to outline how you will conduct your restriction digests as modeled below. Be sure to check the actual concentrations of each restriction enzyme that you will use and adjust the volumes accordingly. Also, each student will have his/her own DNA concentration. Use approximately 1.5 g of DNA in the restriction digest. For many restriction enzymes, 1 Unit of the enzyme will cleave 1 g of DNA at 37 °C for 1 hour in a 50-L reaction mix. The conditions below call for 20 Units of enzyme for every 1.5 g of DNA in a 15-L reaction mix. Table 1. Example of a Theoretical Restriction Digestion Outline* Reaction Mix Contents Ear I 10,000 Units/mL Hae II 20,000 Units/mL EcoR I + Dra I each @ 20,000 Units/mL Vol of Purified DNA (150 g/mL) 10 L 10 L 10 L Vol of Buffer 1.5 L of NEBuffer #1 1.7 L of NEBuffer #4 supplemented with BSA 1.5 L of NEBuffer #4 Vol of dI H2O 1.5 L 2.3 L 1.5 L 1 L of EcoR I Vol of Restriction Enzyme 2 L 1 L 1 L of Dra I Total Reaction Volume 15 L 15 L 15 L *When you generate your own table, insert the appropriate concentrations and volumes everywhere. The data here serve as an example only. 2. Prepare each of the components of the reaction mix as required. The purified DNA should be thawed completely and stored on ice until further use. NEBuffers are commercially available. Each should be fully thawed and stored on ice until further use. Prepare the NEBuffer #4 supplemented with BSA by adding 5 L of 100X BSA


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