1. Which lane or lanes demonstrate partially undigested plasmid DNA? (answer = X
ID: 132264 • Letter: 1
Question
1. Which lane or lanes demonstrate partially undigested plasmid DNA? (answer = X)
2. Which lane or lanes illustrate partially digested DNA? (answer = H)
3. Which lane or lanes illustrate complete digests? (answer = E & X+E )
4. What is the approximate size of the insert? (answer = Approx 8.0 Kbp)
I just want to understand how to approach these questions, i've posted the correct answers but I don't know how they got them! Please and 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
3)Plasmid is a circular DNA. Digestion of plasmid at single site produce 1 linear fragment whereas digestion at 2 sites produce 2 fragments this represent complete digestion of plasmid DNA. Thats why here lane 2and 5 represent complete digestion. Here, the size of plasmid DNA is 11kb. Digestion of plasmid with E+X also give fragments of 11 kb size ( 7000-4000bp).
2) From uncut DNA we represent size of plasmid. Smaller band has size 11 kb. So , plasmid DNA has 11 kb size. If you observe lane 3 , there is 3 band of 5000, 3000 and 2000 ( size calculated from graph) in size means total size is 10 kbp. Therefore lane 3 or H has partially digested DNA.
1) here, X represent approx same pattern as uncut plasmid DNA. It means this lane is partially undigested plasmid DNA.
4) Here, insert represent band at 3.7 mm means size of 8000 bp. So size of insert is 8 kbp.
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