A student cuts an insert and a vector with EcoRI alone…
Contributed by StemPy LabTry it on paper first. A hint, the key idea and the worked solution are here when you need them.
A student cuts an insert and a vector with EcoRI alone. The vector has one EcoRI site. She mixes them and adds ligase.
- How many different kinds of plasmid can form, counting a vector that closes with no insert?
- Name them.
- How can she stop the vector from closing on itself, and what happens to the insert while she does this?
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. Insert and vector cut with one enzyme.
Plan. List the ways in which the ends can join.
Step 1. (a), (b) Both ends of the opened vector are alike, so the vector can close with no insert: one kind. The insert can enter in two directions: two more kinds. That is kinds in all.
Step 2. (c) She treats the opened vector with alkaline phosphatase, which removes the 5 prime phosphates. Ligase needs them, so the vector can not close on itself. The insert keeps its phosphates, so it can still be joined to the vector.
Answer: (a) three. (b) a vector with no insert, an insert in one direction, an insert in the other direction. (c) Alkaline phosphatase on the vector, and the insert is not harmed.
Watch out. The phosphatase is used on the vector only. If it were used on the insert, the insert could not be joined either.
More onBiotechnology
Discussion (0)
Log in to join the discussion.
Loading…