Recombinant dna techniques lab answers
Students were provided with colonies from just one of these recombined products and given the task of identifying the size and orientation of the insert. This was done using alkaline lysis method of plasmid DNA isolation followed by restriction digest and gel elecrophoresis of it.
The insert size and orientation was examined by estimating the fragment sizes using a reference standard. And comparing them with restriction maps of all possible insert configurations. The initial gel was not of a quality sufficient to resolve the fragment size and orientation. Therefore, a model gel is preferentially used for the analysis. Not only did these data reveal the size and orientation of the insert, but they demonstrated some interesting aspects of gel interpretation.
Finally, the productivity of the cloning system was examined via spectrophotometric analyses of the plasmid DNA. Jill comments on the introduction 45s: Sarah describes how she did the research for this report 25s: The in vitro production of proteins continues to burgeon in response to demand for their use in both small-scale exploratory research as well as large-scale therapeutic procedures [1]. Recombinant DNA technology is the platform for protein preparation.
Its application requires experience in a number of baseline molecular biology techniques. This includes the definitive screening and identification of recombined DNA. To this end, the primary aim of this study was for students to learn and practice some of these useful skills. Among the traditional and perpetually valuable tools in synthetic gene construction are the Escherichia coli host cell, restriction enzymes and phagemid cloning vectors.
Each of these is used in this experiment. It yields six fragments when digested with the latter. After synthesis, transformation and cloning of the constructs, it is standard procedure for scientists to analyse their quality and composition.
As discussed, the primary aim of this experiment is to apply and understand something of recombinant DNA technology. Students were therefore provided with both blue and white colony phenotypes that contained just one of the four possible fragments; in just one of its two orientations. Fragment size is estimated by plotting a standard curve of known lengths of linearised DNA kb against their migration distance through the gel matrix mm.
This study does not present any novel ideas. Rather, its role is to assist students in learning and applying generic skills in the field of molecular biology and genetics. Jill comments on the results 31s: Sarah talks about the challenges of writing the results and discussion 1m 18s: The student gel displayed bands of DNA from the blue colony phenotype lanes , the white colony phenotype lanes , and the Hind III reference standard lanes 1, 6 and Standard curve indicating log 10 fragment size against migration, constructed from measurements made from the student gel shown at Figure 6.
Band sizes were estimated using this equation. All digests of the blue colony phenotype yielded single bands of approximately 2.
Two bands were visualized in the lane of undigested DNA. The Bam HI digest of same yielded one band 5. After synthesis, transformation and cloning of the constructs, it is standard procedure for scientists to analyse their quality and composition. As discussed, the primary aim of this experiment is to apply and understand something of recombinant DNA technology. Students were therefore provided with both blue and white colony phenotypes that contained just one of the four possible fragments; in just one of its two orientations.
Fragment size is estimated by plotting a standard curve of known lengths of linearised DNA kb against their migration distance through the gel matrix mm. This study does not present any novel ideas. Rather, its role is to assist students in learning and applying generic skills in the field of molecular biology and genetics.
Jill comments on the results 31s: Sarah talks about the challenges of writing the results and discussion 1m 18s: The student gel displayed bands of DNA from the blue colony phenotype lanes , the white colony phenotype lanes , and the Hind III reference standard lanes 1, 6 and Standard curve indicating log 10 fragment size against migration, constructed from measurements made from the student gel shown at Figure 6.
Band sizes were estimated using this equation. All digests of the blue colony phenotype yielded single bands of approximately 2. Two bands were visualized in the lane of undigested DNA. The Bam HI digest of same yielded one band 5.
Two bands were visualized Refer to next section for description of these Figure 6. Lanes contain DNA from the blue colony phenotype: Lanes show some, but not all expected bands of DNA from The faint almost indistinguishable banding patterns of the student gel were not entirely adequate for completing the required analyses. Therefore, a model gel will also be presented and discussed in this paper. Several bands and smearing were visualized in the lane of undigested DNA.
In the student gel, the experimental concentration of DNA for each of the white and blue colony phenotype samples was The purity of DNA Jill comments on the discussion 22s: Sarah describes how she could have improved her discussion 54s: Restriction mapping of all possible inserts data not shown reveals that the banding pattern of the model gel best resembles that of the Kb insert.
Specifically, the predicted Eco R1 digest bands 4. That these estimates are a little low is explained by the error inherent curve fitting, significant figures and manual measuring of the gel photograph. Gel fitting programs may assist in reducing some of this error.
It could also be that the cells from which this DNA was taken The blue colony phenotype DNA banding patterns were useful and as expected for both the student and model gel. They confirm that these samples do not have the insert present because of the absence of a second Bam HI restriction site.