Cloning OK, it is time to do some subcloning. I want to take My DNA out of plasm

Question

Cloning OK, it is time to do some subcloning. I want to take My DNA out of plasmid A and put it into plasmid B. {A.) My DNA is in plasmid A in the correct orientation. Tell me what enzymes you would use to put it into plasmid B in the correct orientation(5 pts). B. My DNA is in plasmid A in the incorrect orientation. What enzymes would you use to put it into plasmid B in the correct orientation (5 pts) My DNA has only an EcoRI site in it. Sacl and Apal form blunt ends. Alfothers shown produce overhangs. Apal Xhol EcoRI BamH Apal Sacl ECORI Sacl Xhol BamH1 ECORI Plasmid B Plasmid A

Explanation / Answer

Plasmid A has six RE sites namely Apal, Xhol, EcoR1,BamH1, Aplal, SacI and the gene of interest(GoI/'My gene') is placed between EcoR1 and BamH1.

A. If the gene is in the correct orientation the enzymes to be used are Xhol1, BamH1, Poly nucleotide Kinase(PNK) and T4 DNA ligase.

Reasons- 1.as the gene of interest contains an EcoR1 site, we cannot use it for restriction digestion as it will cut the GoI it self. So EcoR1 can be ruled out.

  

3.Now for digestion if Xhol1 and BamH1 are used, in Plasmid A two sticky ends will form; one at the Xhol site and another one at BamH1 site containing the GOI and an EcoR1 site in between two. At this moment PNK is used to make the cohesive ends into blunt ends.

4. As we know that T4 DNA ligase can join blunt ends produced by any restriction enzyme, the insert can easily be put into the space between Sac1 and Xhol1 of Plasmid B.

B. If the GoI is in incorrect orientation i.e in the reverse orientation, there are two enzymes can be used- Restriction enzyme Xhol and T4 DNA ligase. T4 DNA ligase is capable of joining two blunt ends produced by any restriction enzyme.

Now if we use Xhol to digest both Plasmid A and Plasmid B, one cohesive end will be produced at the Xhol sites of both plasmids. if they are allowed to join at this moment, the cohesive Xhol ends will join. Now after purification step if PNK is add the remaining cohesive end will become blunt end. After that if T4 DNA ligase is add, it will join blunt ends puting the GoI('My gene') in correct orientation.

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