A circular vector has 3000 bp. It has an EcoRI site at position 400 and a BamHI site at position…
Contributed by StemPy LabTry it on paper first. A hint, the key idea and the worked solution are here when you need them.
A circular vector has bp. It has an EcoRI site at position and a BamHI site at position . It is cut with both enzymes. An insert of bp has an EcoRI end on the left and a BamHI end on the right. The two are mixed and ligated.
- How long is the stuffer that is lost?
- How long is the backbone of the vector?
- How long is the recombinant plasmid?
- In how many directions can the insert enter?
Need a reminder? The key ideaShow
Cut inside the cloning region of the vector, and not inside the gene. Two different enzymes give directional cloning and stop the vector from closing on itself. Recombinant vector stuffer insert. A diagnostic digest with an off-centre site shows the direction of an insert.
The worked solution
Have you tried it?
Work it out on paper first. It is fine to get stuck or make a mistake: that is how we learn. When you are ready, go through the solution one step at a time, and check each step against your own.
Given. Vector bp; sites at and ; insert bp.
Plan. The piece between the sites is lost. The backbone and the insert make the recombinant.
Step 1. (a) bp.
Step 2. (b) bp.
Step 3. (c) bp.
Step 4. (d) An EcoRI end fits only an EcoRI end, and a BamHI end fits only a BamHI end. So there is one direction.
Answer: bp; bp; bp; one direction.
Check. The recombinant is longer than the vector by the insert minus the stuffer: , and .
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