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Michael Graham Genetics U302 4/11/06 Lab Reports: 8-10

Exercise 8

1.	the expected for plate A was few, while our A plate yielded few. Plate B was expected to yield some colonies but only a few colonies grew. Plate C had no bacterial growth, as was expected. Plate D had a full bacterial lawn, despite only being expected to display a few colonies.

2.	Temperature could certainly factor into transformation efficiency, since the procedure requires the maintenance of specified temperatures in order to transform bacteria. Had the temperature been slightly off in any of the stages where it is stated explicitly, a reduction in efficiency could have resulted. 3.	Adding an antibiotic marker or some other kind of marker could allow for genetic selection, ensuring that only bacteria with a preferred genotype survive on the selection medium. Also, such a gene may be utilized as a tag which can act as a marker to identify which bacteria carry certain genes.

Exercise 9

1.	The purpose of the mini-prep is to isolate plasmid DNA in the transformed E.coli and verify that the phenotype displayed is due to the incorporation of the plasmid into the bacterial genome. 2.	DNA is separated from other cellular materials through alkaline lysis in this case. This separates DNA from other cellular debris. 3.	Genomic DNA is separated from genomic DNA in this procedure through thanks to the addition of the SDS/NaOH solution to the tube containing the genetic material. The SDS/NaOH lyses cells and dissolves other organic materials; after a five minute wait, and a mixing of the solutions by rapid but gentle tube-inversion, the genomic DNA becomes dissolved in a precipitate along with other cellular materials. This precipitate can then be pelleted using a microfuge. The pelletting causes the precipitate to condense and allows for facilitated extraction of the plasmid DNA from the supernatant fluid. 4.	Results could be influenced heavily be the amount of care put into the procedure: the solution must several times be mixed in a careful manner, usually very gently. Failing to follow this guideline could result in a skewing of results because the genomic DNA could fracture into smaller pieces that do not easily separate from the plasmid DNA in solution. Additionally, temperature plays an important role and failing to regulate it appropriately could also result in yield alteration.

Exercise 10

1. Although it is difficult to estimated sizes due to poor sketch quality for the experiment as it appeared in class, based on the drawing it appears that at least 4 markers are larger than 1650. Most of the markers, however, are likely less than this, as many of them appear quite thin. 3. Most of the mini-prep DNA on the strip is generally smaller and there are significantly fewer strips in the mini-prep lane. Also, two bands appear in column C that are cut in half vertically. This has probably occurred due to the presence of enzymes in which cleave the plasmid DNA in half. 4. According to the ideal gel diagram, there should be few colonies in the A Lane which we see with the appearance of only 2 bands. Lane B has a number of markers, which is only partially incongruous with the expected for an ideal gel, which says there should be some bands. The ideal gel expects none for Lane C but our lane does not comply and has a number of bands within it. Lane D should receive a full lawn, but for this case only has 2 bands.