RESTRICTION ENZYMES. HHMI Experiment to follow Wheat Germ Extraction (modified by Donald Holmquist, 2001) In this exercise you will; 1. Digest two plasmids with EcoRI 2. 3. 4. Recombinant DNA technology or “gene splicing,” as it is frequently called, often involves insertion of selected DNA sequences from a variety of sources into plasmids which are then transformed into bacteria. This process would not be possible without accurate and predictable ways of cutting the DNA of the plasmid and the foreign source. Usually, restriction enzymes only cut the DNA at or near a very specific nucleotide sequence known as a recognition site. Some restriction enzymes cut both sides of the helix at the same point. This type of asymetric cut leaves a single stranded sequence hanging on either 5’ or 3’ end. Procedure: A. (note- all components involved in digestion should be kept on ice) 1. 14μl distilled water 2μl 10 X EcoRI Buffer 3μl of pUC19 DNA (uncut) lμl of Restriction enzyme EcoRI 2. 3.
B. 1. 2μl 10 X EcoRI buffer 2. 3. C. 1. How to Graph with Microsoft Excel. How to Graph with Microsoft Excel Spectrophotometry Laboratory 1. Open Microscoft Excel Graphs 1-4 ( Blue and Yellow solutions ) Absorption Spectrum Graphs ( 4) A boxes independent variable - wave length B boxes dependent variables - % transmittance and absorbance ( O.D.) Skip Box A1 in Column A In box A2 begin to enter your data Start with the wavelength of 420 nm and go to 620 nm In column B, box, B1 write absorbance or % T Enter data into B column Select columns A and B Click on chart icon Select Line Graph - This is the choice because you have a correlation between the two variables an important relationship betwen the light passing through the solution and % transmittance and absorbance Go to chart options Make a title for your graph Choose labels for each axis Go to finish To edit Graph use the pull down menu.
For instance if you want gridlines, select Gridlines - it will give you a grid for major and minor axes You can change the position of the Legend by selecting Legend Graphs 5-8 Repeat. Size of DNA Molecules Determined by Agarose Gel Electrophoresis. Laboratory Exercise. Laboratory Exercise: ELECTROPHORESIS & MIGRATION DISTANCE In order to analyze the products of digestion by restriction enzymes, it is necessary to visualize the fragments produced. Gel electrophoresis is a technology that has been developed to separate DNA fragments, taking advantage of the inherent chemistry of the DNA molecule.
DNA is negatively charged due to the phosphate groups embedded in side rails of the helix. When DNA is placed in an electric field, the fragments will migrate toward the positive pole (anode) as they are being repelled from the negative pole (cathode). The sorting of fragment size takes place in an agarose gel. The agarose gel is placed in an electrophoresis chamber and covered with a buffer solution containing ions that help pass the current through the gel. Following electrophoresis, the gel is soaked in a stain that will diffuse throughout the gel and become concentrated in areas where it is binding to DNA fragments. In this lab you will: · Learn to pour a gel.
Laboratory Exercise. Laboratory Exercise: ISOLATION OF DNA FROM WHEAT GERM In this lab, you will: Isolate DNA from a eukaryotic source Run gel electrophoresis to visualize the DNA Optionally cut the genomic DNA using the restriction enzyme EcoR1 All cells contain nucleic acids. Bacterial cells have no nucleus and therefore have their DNA unbounded by a nuclear membrane. Wheat Germ DNA Isolation Procedure: Gather all materials needed for the extraction Prepare solutions Weigh ingredients Become familiar with the lab protocol a.
B. C. D. A. B. C. D. E. A. B. (link to elecgtrophoresis/migration distance lab for gel instruction, and omit steps c-g) c. D. E. F. G. (to destain - put 100 ml deionized/distilled water into a destaining tray. (link to electrtophoresis/migration distance lab for photography instruction-omit steps if linked) a. B. C. D. E. F. Laboratory Exercise. Laboratory Exercise-BACTERIAL TRANSFORMATION HHMI Laboratory for Honors Genetics (modified by Donald Holmquist ,2001) In this exercise you will: 1. Prepare competent E. coli cells for transformation. 2. 3. Introduction: Relatively simple organisms, like E. coli, other bacteria and yeasts, often contain circular DNA molecules called plasmids. Bacteria are capable of plasmid uptake through the process of bacterial transformation. This assay for inserted fragments is known as alpha-complementation.
When, however, a large fragment of foreign DNA is inserted into the polylinker of pUC19, this extra DNA causes such a great disruption of the plasmid-encoded alpha subunit that it no longer has activity and cannot complement the subunit encoded in the cell's genome. Procedure (each group) A. 1. 2. 3. 4. 5. 6. 7. B. 1. 2. 3. 4. 5. 6. 7. 8. C. 1. 2. 3. 4. D. 1. 2. E. 1. 2. 3. 4. 5. 6. 7. 8. 1. 2. 3. 4. A. 1. 2. 3. B. 1. 2. 3. 4. Laboratory Techniques. Laboratory Techniques.