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This outstanding program provides a solid foundation in the molecular biology of DNA. Your students will investigate such contemporary topics as restriction nuclease mapping, the complexity of the prokaryotic and eukaryotic genomes and the nucleosome structure of the chromosome. The studies on plasmid amplification using recombinant DNA techniques enable students to conduct the same procedures that form the basis of the biotechnology industry. 301. The Length of DNA Molecules Electrophoresis in agarose gels is the most common method used for determining the size of DNA molecules. In this introductory exercise, students determine the length of an unknown DNA molecule by comparing its electrophoretic mobility with six DNA molecules of known size as shown below. 302. Restriction Nuclease Mapping of DNA Bacteriophage lambda is a DNA virus that attacks E. coli. Here, students dissect lambda DNA using the restriction endonucleases EcoR1 and BamH1 in order to identify specific sites, sequences, and structures along the phage genome. This single exercise enables students to explore a number of exciting topics in molecular biology, including the specificity of restriction endonucleases, DNA mapping strategies, complementary base-pairing of DNA, and the structure of a viral genome.
Length of DNA EXP-301 1 2 3 4 5 6 7 8
DNA fragments of known lengths (lanes 1, 3, 5, and 7) and unknown length (lanes 2, 4, 6, 8) are separated by electrophoresis
Restriction Nuclease Mapping EXP-302 1 2 3 4 5 6 7 8
Lambda phage DNA was digested with EcoR1 or EcoR1 plus BamH1 prior to this electrophoretic separation.
DNA STAINING PROCEDURE Our Programs now use a simple two-step procedure for staining DNA in agarose gels. First, a nontoxic purple dye is incorporated into the agarose gel prior to electrophoresis. Faint purple DNA bands can be clearly seen after 30 minutes of electrophoresis. This rapid procedure enables the student to obtain preliminary results of the experiment in a short period of time. After the electrophoretic run, gels are incubated with methylene blue to complete the staining procedure. 303. Plasmid DNA Structure Plasmids are small circular DNA molecules found in most bacteria. A plasmid can exist in different structural states and these states can be distinguished by their migration on agarose gels. In this experiment, students study these structures using enzymes and electrophoresis and show that the structures are interconvertible. This exercise provides an introduction to higher-order DNA structure which is thought to be important in the control of transcription and replication in bacteria and, perhaps, in higher organisms as well. 304. Molecular Cloning This two part exercise provides state-of-the-art information and practical experience with a variety of techniques that form the foundation of the biotechnology industry. In part A, students create a strain of E. coli that is resistant to the antibiotic ampicillin by introducing a plasmid that contains an ampicillin-resistance gene. The success of the transformation is monitored by growing the bacteria on an ampicillin-containing media. This experiment provides sufficient sterile materials for sixteen platings. In Part B, students isolate the amplified plasmid from the bacteria without the use of toxic chemicals. Then they digest the isolated DNA with EcoR1 and compare the electrophoretic properties of linear and circular plasmid DNA molecules. By inserting desired DNA segments into plasmids, this procedure has enabled scientists to amplify more than 1000 specific genes including those for human interferon, insulin, and growth hormone. Part B of the exercise requires a centrifuge that can be operated at a force of at least 3,000 x g (such as a microcentrifuge or a larger floor model). The entire exercise requires three 2-3 hour laboratory periods and the materials provided are shown below. Materials for EXP 304
305. Identifying Satellite Sequences A large fraction of the DNA of vertebrates consists of nucleotide sequences that are repeated thousands or more times in the genome. Satellite sequences are a major class of these repeated elements and these sequences are found in most eukaryotic organisms. There are about 1 million cutting sites for the restriction enzyme EcoRI in the typical vertebrate genome and about 1 million DNA fragments are generated following digestion of vertebrate DNA with this enzyme. When this DNA is separated by electrophoresis, the large number of differently-sized fragments appear as a background smear on the gel lane. However, as shown in the figure below, DNA fragments from the highly repeated satellites form discrete bands because they are the same size. In this exercise, students identify satellite sequences in the cow genome and determine if such sequences are present in chicken DNA. Satellite DNA EXP-305 1 2 3 4 5 6 7 8
DNA from calf thymus (even numbered lanes) was digested with EcoR1 prior to this electrophoretic separation. Note the discrete satellite DNA band.
306. The Nucleosome Structure of Chromatin The primary level of chromosome structure in eukaryotes occurs when the DNA molecule is wrapped around histone proteins into particles called nucleosomes. Evidence for this "beads on a string" model is derived from nuclease digestion studies. When nuclei are incubated with micrococcal nuclease, the enzyme cleaves the linker DNA between nucleosomes (the string) but not the nucleosomal core DNA (the beads). When the DNA isolated from these nuclei is electrophoresed on agarose gels, the DNA exhibits a ladder pattern with the length of the DNA in each band representing a multiple of the nucleosomal unit as shown in the Figure below. With this background information, students first examine nucleosomal DNA from calf thymus that is given in this chemical package. They then prepare nuclei from wheat germ, incubate them with micrococcal nuclease and examine the ladder pattern in the DNA by electrophoresis. This exercise requires two 2-3 hour laboratory periods. Nucleosome DNA EXP-3061 2 3 4 5 6 7 8
Note the ladder pattern produced when the DNA in chromatin is cleaved by micrococcal nucleases (lanes 2-4, 6-8).
Price List - Standard Laboratory Program 3
Individual Experiments Each of the individual experiments is supplied with the chemicals and laboratory guides needed for 16 students working in pairs. If you chose one or more of the experiments below, you should also order Electrophoresis Package 3/4. Electrophoresis Package 3/4 provides sufficient agarose, gel stains and electrophoresis buffers for up to 6 of the individual experiments in this series. The package also includes the dye for rapid DNA staining during electrophoresis (See above). Electrophoresis Package 3/4 is also suitable for the experiments in Standard Laboratory Programs 4 and 10 as well as a majority of our IND series.
The following experiments include Chemicals and Instructions.
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