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Ex_4_polytene_09 - Exercise 4 Chromosomes mitosis and...

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Exercise 4. Chromosomes, mitosis, and meiosis 53 EXERCISE 4 ANALYSIS OF THE RESTRICTION DIGEST POLYTENE CHROMOSOMES, MITOSIS, MEIOSIS, AND CYTOKINESIS “I am a firm believer that without speculation there is no good and original observation” Charles Darwin (1809-1882) * AGAROSE GEL ELECTROPHORESIS OF DNA * THE STEREO (DISSECTING) MICROSCOPE * DROSOPHILA POLYTENE CHROMOSOMES * NUCLEAR AND CELL DIVISION * POLLINATION OF F1 PLANTS Equipment and supplies needed restriction digests from last time electrophoresis chambers agarose balance, weighing pans and spoons gel buffer (0.5xTBE) microwave oven/padded gloves graduated cylinders Erlenmeyer flasks P20 pipetman and tips microfuge racks power supplies Dye = tracking dye solution ice containers dilute ªDNA (0.1μg/μl) ice container heat block 100°C vortex mixer microcentrifuge ethidium bromide image analyzer computers and Cricket Graph ruler Polytene Chromosomes dissection microscopes B. rapa flowers slides and coverslips compound microscopes aceto-orcein stain Drosophila larvae 0.7% NaCl in squirt bottles kim wipes immersion oil fine forceps small petri dishes prepared slides of polytene chromosomes lens paper Pasteur pipettes Mitosis and Meiosis Prepared slides of plant root Prepared slides of fish blastula Pollination of F1 Plants Dried Bees F1 Plants support sticks plant ties
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Exercise 4. Chromosomes, mitosis, and meiosis 54 The purpose of this exercise is to analyze the restriction fragments of lambda chromsomomes by agarose gel electrophoresis, and to prepare and examine giant chromosomes of the fruit fly, Drosophila. PART I: Analysis of the Restriction Digest Agarose Gel Electrophoresis is the most common way of separating and analyzing fragments of DNA, such as we produced last week in our restriction digest of the lambda bacteriophage genome. Electrophoresis is a process whereby charged molecules are separated in an electric field. The rate at which molecules move in an electric field, their so-called mobility, differs depending on the size, shape, shape and charge of the individual molecules. Separation of molecules during electrophoresis, therefore, occurs due to differences in the mobility of molecules through a medium Large weakly charged molecules will move slowly in an electric field (i.e. have a low mobility), whereas small highly-charged molecules will move more quickly. If both kinds of molecules are present in a mixture, then they can be physically separated by electrophoresis. Because different DNA molecules are chemically and structurally very similar to each other, mobility of different DNAs is largely determined by the length of the pieces of DNA. Electrophoresis through agarose is commonly used to separate DNAs of intermediate to large size (100 to several thousand base pairs). Agarose is a complex, branched polysaccharide extracted from seaweeds. When it is heated up, agarose molecules become fully extended and dissolve in aqueous solutions. When cooled, however, agarose molecules fold up on themselves and gel into meshwork that is like a 3-dimentional fisherman’s net.
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