B BIO/CHEM CLAss ACTIVITY 6 MOLECULAR ULUlul U. 375 IN You purify DNA from the i

Question

B BIO/CHEM CLAss ACTIVITY 6 MOLECULAR ULUlul U. 375 IN You purify DNA from the individual colonies and perform a restriction digest to completion using You run the results on an agarose gel Hind (iii) Lane 1 is a DNA sample of the vector with no insert. Lane 2 is a DNA sample with the with the insert. Draw map of the vector with the insert, Xbal labeling the of the restriction sites for EcoRI, Hindlll and and how far apart the sites are from each other. DNA size 5700 bp 7000 bp 6000 bp 5000 bp 5000 bp 4000 bp 3000 bp 2000 bp 00 1000 bp 300 bp 500 bp (iv) You realize that the insert can be ligated into the vector in a different pattern orientation. Using all the given information, fill in the predicted banding has an on the agarose gel in lane 3 for a Hindlll-digested DNA sample that the insert ligated in a different orientation from lane 2. Label the size of DNA band. (v) How could you prevent the insert ligating in two different orientations

Explanation / Answer

you have digested the vector with HindIII and got a single band of 5000bp.

lane 2 contain two bands 5700 and 300bp. from above given information it is clear that vector is 5000bp and insert is 1000bp. since we got two bands which mean that insert DNA also contains a restriction site for HindIII. now if we ligate that then we got a circular vector + insert DNA molecule which is 6000bp.

since you have not provided the information regarding whether the other RE site is present in insert too. also you did not told that whether these sites are present in MCS region or not. so I am combining the three RE site together and making a DNA sequence.

5' TCTAGA ATTC AAGCTT 3' = first 6 bp= XbaI, bold 6 bp= EcoRI, and last 6 bp= HindIII.

now if insert is ligated into vector in different orientation then we will get two bands of 5300 bp and 700 bp.

with the use of alkaline phosphatase enzyme we will be able to prevent insert ligation in different orientation.

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