Given that all the digests were done with 0.1?g of DNA, how much DNA is probably
ID: 132272 • Letter: G
Question
Given that all the digests were done with 0.1?g of DNA, how much DNA is probably represented by the band with a migration distance of 6.8 mm in lane “X + E”?
Answer = Approx. 40ng
10. Which band in lane “U probably represents supercoiled plasmid DNA?
Answer = 3.0 band
please explain how they got these answers!! thank you
A recombinant plasmid was generated as follows: PCR was used to amplify a region of the yeast genome with a pair of "Forward" and "Reverse" primers which had the restriction sites Pst and Pvu Il added to their 5' ends. Following digestion of the amplicon with Pst I and Pvu lI, it was ligated into the pUC vector digested with Pst I and Sma l. Pst I Pvu lI Sma I 5..CTGCAG..3 5CAGCTG...3 GTCGAC... 5 3 GACGTC..5 To verify the identity of the recombinant plasmid, restriction digests followed by agarose gel electrophoresis were performed. The gel and the corresponding standard curve of the molecular size markers are illustrated. The values next to each band indicate the migration distances in mm. 2.02.1 2.1 3.03.7 U: Uncut E: EcoRI H: HindIII X: XhoI _5.4 ? ?6.8 10 Migration distance (mm)Explanation / Answer
To estimate the concentration of DNA from gel run, we need the concentration chart provided by the manufacturer. Usually, a when a generuler or marker is bought, it is provided with a chart, which shows the different bands obtained upon running the marker and their sizes. Each band would have different intensity. Each intensity corresponds to a specific concentration of DNA.
We usually compare the intensity of our DNA with that of the bands in the marker lane to estimate the concentration. Similarly, the 6.8 mm band in X + E lane might have corresponded to the intensity of 40 ng band.
When an uncut plasmid is run on gel, usually, it produces 3 bands. The plasmid would be in 3 conformations, supercoiled, circular and linear. Supercoiled state of plasmid moves faster than the other. Therefore, the fastest migrating band or the most migrated band corresponds to supercoiled plasmid.
In the given figure, since the band at 3mm distance is fast migrating, it would be supercoiled.
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