QUESTION AND ANSWER - PLEASE EXPLAIN HOW THEY GOT THIS ANSWER!! All I need is to
ID: 132268 • Letter: Q
Question
QUESTION AND ANSWER - PLEASE EXPLAIN HOW THEY GOT THIS ANSWER!! All I need is to uNDERSTAND!
1. The PCR product inserted in the pUC vector was used as a probe for a Southern analysis. Which bands in lane H would hybridize to the probe? (A = all of them)
2. How many times does “H” cut within the insert? (A = twice)
3. What fragment size(s) would be predicted from a digest of the recombinant plasmid with Pst I and SmaI? (A = 11kbp)
8. What is the probable distance from the Eco RI going towards the insert to the Xho I restriction site? (A = 4.0 or 7.0 Kbp)
Please and thank you!
UPDATED: size od band with migration distance of 2 is 11kbp, as derived from the plotted graph.
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
1) the probe will be hybridized by all the enzyme as we can see all three bands corresponding to H well. EcoR1 HIND III Xho1 all three can hybridized the probe.
2) yes it can cut twice because the insertion has to made in two places
3) fragment size would be size of each fragment +1
=6+6-1=11
4) it would be 4 or 7 as the total was 11 kbp and the enzyme has to go at least 3bp to reach the target Xho1 site.