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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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Mapping gel (5% 29:1 polyacrylamide, 1 × TBE) separating restriction enzyme fragments of plasmid DNA containing a 2.6-kb insert of wild-type D. melanogaster DNA from the dusky area of the genome. Aliquots of the same digests separated on the agarose gel (Figure 3) were separated by nondenaturing polyacrylamide minigel electrophoresis. Only the gel with digests of the plasmid with the insert with orientation A is shown. DNA in these 1-mm-thick gels was stained with ethidium bromide, destained in 1 × TBE, and visualized and digitally photographed as described above (Figure 3). Migration of standard marker fragments (M; MspI-digested pBR322) was measured (using the ruler pictured or an external one), and each student constructed a standard curve for the gel. The refined resolution and extrapolated sizes of the small fragments (<600 bp) confirmed fragments detected on the agarose gel as well as helped detect doublets and very small fragments such as the BamHI-digested fragments at approximately 70 bp. This photograph (made available online for student use) was enhanced (contrast and brightness) using Adobe Photoshop.
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Figure 4: Mapping gel (5% 29:1 polyacrylamide, 1 × TBE) separating restriction enzyme fragments of plasmid DNA containing a 2.6-kb insert of wild-type D. melanogaster DNA from the dusky area of the genome. Aliquots of the same digests separated on the agarose gel (Figure 3) were separated by nondenaturing polyacrylamide minigel electrophoresis. Only the gel with digests of the plasmid with the insert with orientation A is shown. DNA in these 1-mm-thick gels was stained with ethidium bromide, destained in 1 × TBE, and visualized and digitally photographed as described above (Figure 3). Migration of standard marker fragments (M; MspI-digested pBR322) was measured (using the ruler pictured or an external one), and each student constructed a standard curve for the gel. The refined resolution and extrapolated sizes of the small fragments (<600 bp) confirmed fragments detected on the agarose gel as well as helped detect doublets and very small fragments such as the BamHI-digested fragments at approximately 70 bp. This photograph (made available online for student use) was enhanced (contrast and brightness) using Adobe Photoshop.

Mentions: Agarose is limited at resolving small (<600 bp) fragments that are close in size, so about a third of each mapping digest is size separated through polyacrylamide minigels, one gel for each orientation (Figure 4 and unpublished data). Digital photographs of these gels are made available to the students on the Internet and with class handouts. As expected, the polyacrylamide gels do resolve different-sized fragments that migrate as one band on the agarose gel (e.g., Figure 4 and Figure 3, B/P digest for orientation A); however, as warned by Sambrook et al. (1989), the polyacrylamide also causes one of the smaller fragments to migrate relatively faster than its size should allow (compare Figure 3, V/P digest, with Figure 4, V/P digest). Such an inconsistency in migration is unusual, but it can be used to teach a valuable lesson to the students: They should check data from different protocols for consistency, and if aberrancy is found, they should pursue a logical explanation and not just assume that they must have done something wrong.


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)

Mapping gel (5% 29:1 polyacrylamide, 1 × TBE) separating restriction enzyme fragments of plasmid DNA containing a 2.6-kb insert of wild-type D. melanogaster DNA from the dusky area of the genome. Aliquots of the same digests separated on the agarose gel (Figure 3) were separated by nondenaturing polyacrylamide minigel electrophoresis. Only the gel with digests of the plasmid with the insert with orientation A is shown. DNA in these 1-mm-thick gels was stained with ethidium bromide, destained in 1 × TBE, and visualized and digitally photographed as described above (Figure 3). Migration of standard marker fragments (M; MspI-digested pBR322) was measured (using the ruler pictured or an external one), and each student constructed a standard curve for the gel. The refined resolution and extrapolated sizes of the small fragments (<600 bp) confirmed fragments detected on the agarose gel as well as helped detect doublets and very small fragments such as the BamHI-digested fragments at approximately 70 bp. This photograph (made available online for student use) was enhanced (contrast and brightness) using Adobe Photoshop.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046893&req=5

Figure 4: Mapping gel (5% 29:1 polyacrylamide, 1 × TBE) separating restriction enzyme fragments of plasmid DNA containing a 2.6-kb insert of wild-type D. melanogaster DNA from the dusky area of the genome. Aliquots of the same digests separated on the agarose gel (Figure 3) were separated by nondenaturing polyacrylamide minigel electrophoresis. Only the gel with digests of the plasmid with the insert with orientation A is shown. DNA in these 1-mm-thick gels was stained with ethidium bromide, destained in 1 × TBE, and visualized and digitally photographed as described above (Figure 3). Migration of standard marker fragments (M; MspI-digested pBR322) was measured (using the ruler pictured or an external one), and each student constructed a standard curve for the gel. The refined resolution and extrapolated sizes of the small fragments (<600 bp) confirmed fragments detected on the agarose gel as well as helped detect doublets and very small fragments such as the BamHI-digested fragments at approximately 70 bp. This photograph (made available online for student use) was enhanced (contrast and brightness) using Adobe Photoshop.
Mentions: Agarose is limited at resolving small (<600 bp) fragments that are close in size, so about a third of each mapping digest is size separated through polyacrylamide minigels, one gel for each orientation (Figure 4 and unpublished data). Digital photographs of these gels are made available to the students on the Internet and with class handouts. As expected, the polyacrylamide gels do resolve different-sized fragments that migrate as one band on the agarose gel (e.g., Figure 4 and Figure 3, B/P digest for orientation A); however, as warned by Sambrook et al. (1989), the polyacrylamide also causes one of the smaller fragments to migrate relatively faster than its size should allow (compare Figure 3, V/P digest, with Figure 4, V/P digest). Such an inconsistency in migration is unusual, but it can be used to teach a valuable lesson to the students: They should check data from different protocols for consistency, and if aberrancy is found, they should pursue a logical explanation and not just assume that they must have done something wrong.

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