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A semester-long project for teaching basic techniques in molecular biology such as restriction fragment length polymorphism analysis to undergraduate and graduate students.

DiBartolomeis SM - CBE Life Sci Educ (2011)

Bottom Line: Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation.Finally, results from everyone in the class are required for the final analysis.Results of pre- and postquizzes and surveys indicate that student knowledge of appropriate topics and skills increased significantly, students felt more confident in the laboratory, and students found the laboratory project interesting and challenging.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Millersville University, Millersville, PA 17551-0302, USA. sdibarto@millersville.edu

ABSTRACT
Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation. I describe a 12-wk laboratory project suitable for upper-level undergraduates and first-year graduate students, in which the students molecularly locate and map a gene from Drosophila melanogaster called dusky and one of dusky's mutant alleles. The mapping strategy uses restriction fragment length polymorphism analysis; hence, students perform most of the basic techniques of molecular biology (DNA isolation, restriction enzyme digestion and mapping, plasmid vector subcloning, agarose and polyacrylamide gel electrophoresis, DNA labeling, and Southern hybridization) toward the single goal of characterizing dusky and the mutant allele dusky(73). Students work as individuals, pairs, or in groups of up to four students. Some exercises require multitasking and collaboration between groups. Finally, results from everyone in the class are required for the final analysis. Results of pre- and postquizzes and surveys indicate that student knowledge of appropriate topics and skills increased significantly, students felt more confident in the laboratory, and students found the laboratory project interesting and challenging. Former students report that the lab was useful in their careers.

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Southern hybridizations showing RFLPs between restriction enzyme–digested Canton S (CS) and dusky73 (dy73) genomic DNAs. The gel shown in Figure 5 was sliced in two and blotted onto nylon membranes, hybridized with biotinylated plasmids containing a 2.6-kb EcoRI-digested fragment of the CS genome, and detected with streptavidin–alkaline phosphatase conjugate, BCIP, and NBT. The blots (mirror images of the gels due to the blotting procedure) were paired up and digitally photographed (Nikon Coolpix995 camera). This photograph (which was posted online for student use) was enhanced (autolevels, contrast, brightness, and annotation) with Photoshop. The standard marker (M) is a mixture of biotinylated HindIII-digested lambda DNA and SalI-digested lambda DNA that had been size separated and blotted with the 1.25-μg HindIII-digested lambda DNA visible on the EtBr-stained gel (Figure 5).
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Figure 6: Southern hybridizations showing RFLPs between restriction enzyme–digested Canton S (CS) and dusky73 (dy73) genomic DNAs. The gel shown in Figure 5 was sliced in two and blotted onto nylon membranes, hybridized with biotinylated plasmids containing a 2.6-kb EcoRI-digested fragment of the CS genome, and detected with streptavidin–alkaline phosphatase conjugate, BCIP, and NBT. The blots (mirror images of the gels due to the blotting procedure) were paired up and digitally photographed (Nikon Coolpix995 camera). This photograph (which was posted online for student use) was enhanced (autolevels, contrast, brightness, and annotation) with Photoshop. The standard marker (M) is a mixture of biotinylated HindIII-digested lambda DNA and SalI-digested lambda DNA that had been size separated and blotted with the 1.25-μg HindIII-digested lambda DNA visible on the EtBr-stained gel (Figure 5).

Mentions: In this project, every effort is made to have each student work in as small a group that is practical for the particular procedure being done. With this in mind, each gel that is to be blotted is cut in half, resulting in six Southern blots. In a “real” lab, the gels would remain intact; however, splitting the gels allows up to only four students per group, greatly increasing the chance of hands-on experience for each student. Once the blots are hybridized and their probes detected, then the blots are paired to represent the full gels (Figure 6).


A semester-long project for teaching basic techniques in molecular biology such as restriction fragment length polymorphism analysis to undergraduate and graduate students.

DiBartolomeis SM - CBE Life Sci Educ (2011)

Southern hybridizations showing RFLPs between restriction enzyme–digested Canton S (CS) and dusky73 (dy73) genomic DNAs. The gel shown in Figure 5 was sliced in two and blotted onto nylon membranes, hybridized with biotinylated plasmids containing a 2.6-kb EcoRI-digested fragment of the CS genome, and detected with streptavidin–alkaline phosphatase conjugate, BCIP, and NBT. The blots (mirror images of the gels due to the blotting procedure) were paired up and digitally photographed (Nikon Coolpix995 camera). This photograph (which was posted online for student use) was enhanced (autolevels, contrast, brightness, and annotation) with Photoshop. The standard marker (M) is a mixture of biotinylated HindIII-digested lambda DNA and SalI-digested lambda DNA that had been size separated and blotted with the 1.25-μg HindIII-digested lambda DNA visible on the EtBr-stained gel (Figure 5).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046893&req=5

Figure 6: Southern hybridizations showing RFLPs between restriction enzyme–digested Canton S (CS) and dusky73 (dy73) genomic DNAs. The gel shown in Figure 5 was sliced in two and blotted onto nylon membranes, hybridized with biotinylated plasmids containing a 2.6-kb EcoRI-digested fragment of the CS genome, and detected with streptavidin–alkaline phosphatase conjugate, BCIP, and NBT. The blots (mirror images of the gels due to the blotting procedure) were paired up and digitally photographed (Nikon Coolpix995 camera). This photograph (which was posted online for student use) was enhanced (autolevels, contrast, brightness, and annotation) with Photoshop. The standard marker (M) is a mixture of biotinylated HindIII-digested lambda DNA and SalI-digested lambda DNA that had been size separated and blotted with the 1.25-μg HindIII-digested lambda DNA visible on the EtBr-stained gel (Figure 5).
Mentions: In this project, every effort is made to have each student work in as small a group that is practical for the particular procedure being done. With this in mind, each gel that is to be blotted is cut in half, resulting in six Southern blots. In a “real” lab, the gels would remain intact; however, splitting the gels allows up to only four students per group, greatly increasing the chance of hands-on experience for each student. Once the blots are hybridized and their probes detected, then the blots are paired to represent the full gels (Figure 6).

Bottom Line: Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation.Finally, results from everyone in the class are required for the final analysis.Results of pre- and postquizzes and surveys indicate that student knowledge of appropriate topics and skills increased significantly, students felt more confident in the laboratory, and students found the laboratory project interesting and challenging.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Millersville University, Millersville, PA 17551-0302, USA. sdibarto@millersville.edu

ABSTRACT
Several reports on science education suggest that students at all levels learn better if they are immersed in a project that is long term, yielding results that require analysis and interpretation. I describe a 12-wk laboratory project suitable for upper-level undergraduates and first-year graduate students, in which the students molecularly locate and map a gene from Drosophila melanogaster called dusky and one of dusky's mutant alleles. The mapping strategy uses restriction fragment length polymorphism analysis; hence, students perform most of the basic techniques of molecular biology (DNA isolation, restriction enzyme digestion and mapping, plasmid vector subcloning, agarose and polyacrylamide gel electrophoresis, DNA labeling, and Southern hybridization) toward the single goal of characterizing dusky and the mutant allele dusky(73). Students work as individuals, pairs, or in groups of up to four students. Some exercises require multitasking and collaboration between groups. Finally, results from everyone in the class are required for the final analysis. Results of pre- and postquizzes and surveys indicate that student knowledge of appropriate topics and skills increased significantly, students felt more confident in the laboratory, and students found the laboratory project interesting and challenging. Former students report that the lab was useful in their careers.

Show MeSH