Expt 1 Report Qs and Answer PDF

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Experiment 1 Report Qs and Answers...

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1. From the facsimile copy of the gel results, draw a standard curve of log10[kb pairs] versus distance from the origin wells in millimeters (mm) for the DNA linear standards on normal scale graph paper. Or, plot kb pairs versus distance migrated on semi-log graph paper. You must include your data table of distances and standards. Use either plot to determine the molecular size of plasmid pUC18 in kilobase or base pairs. [Note: Compare the number of standards listed in the Result Sheet and Lab Manual to the number of bands seen in Lane 3 in the gel, which depicts only 7 bands.] [1 mark]

2, 2nd band: 38mm log10(kb) = 0.44 Molecular size of pUC18 = 2.75 kb pairs = 2750 bp

2. You are given two plasmids of different sizes; namely, 4 kb and 6 kb. Each is in a different conformation, that is, one is linear, one supercoiled, but you do not know which is which. Devise a simple method of determining which is which, using only the tools available from Experiment 1 (standards; agarose gel). [1 mark] [Clue: Do the same as Expt 1, that is, gel and size estimations, and explain the results you should see in the gel for both plasmids.] The plasmid samples containing gel-red dye, 40% sucrose and 100mM EDTA, are run in an agarose gel electrophoresis against DNA linear standards of known size in a lane. Construct a standard curve of the standard samples against migrated distance, this is used to estimate the migrated distance of the two plasmid samples. The 6 kb plasmid should be a linear conformation indicated by a shorter distance travelled, as its length and flexibility causes it to bump into the walls of the pores, slowing the migration rate. The 4 kb plasmid should be a supercoiled conformation indicated by a further migration as it is tightly constricted and will not bump into the pore’s walls, thus migrating faster.

3. In this experiment, the DNA was stained by Gel-Red, already present throughout the cast agarose gel. Say we decided to do it differently and an aliquot of Gel-Red was added to the DNA samples, but not in the gel, as was the case in this experiment. Would the experiment still work? Could you still run the samples and see the DNA in the gel without the Gel-Red present in the gel? Explain. [1mark] [Clue: A matter of if and when Gel-Red binds to DNA.] Yes the experiment would still work and DNA bands can be seen as Gel-Red will bind via intercalation between base pairs. The structures of DNA and Gel-Red dye have similar planar rings, allowing the dye to stack in between DNA base pairs, therefore will fluoresce in UV light. However the experiment will take longer because dye bound to DNA causes it to swell, making it more difficult to move through the gel.

4. In your estimation of the plasmid size in this experiment, you used the Result Sheet to plot the linear standards. The sheet depicts only 7 standards in the gel photo, yet the information above and on the Result Sheet (point ‘2’) lists 8 standards. Explain this apparent anomaly. [1 mark] [Clue: 8 into 7 will not go.] This may have occurred due to the 8th band in results being too faint to be seen...