A laboratory plans five jobs. Say which method it should use to get the recombinant DNA into the…
Contributed by StemPy LabTry it on paper first. A hint, the key idea and the worked solution are here when you need them.
A laboratory plans five jobs. Say which method it should use to get the recombinant DNA into the host.
- Into Escherichia coli.
(ii) Directly into the nucleus of a mouse cell.
(iii) Into wheat cells grown in a dish, with gold particles.
(iv) Into the cells of a tobacco leaf, with a changed bacterium that carries a Ti plasmid.
- Into human cells, with a changed virus.
Then answer: a foreign gene is ligated into the PstI site that lies inside ampR of pBR322. On which plates will the recombinants grow?
Need a reminder? The key ideaShow
The selectable marker finds the transformants. Insertional inactivation finds the recombinants. A foreign gene in the BamHI site of tetR gives colonies that grow on ampicillin and not on tetracycline. In a lacZ vector, white colonies are the recombinants.
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. Five hosts, and a gene in the PstI site of ampR.
Plan. Match the method to the host.
Step 1. (i) Competent cells made with calcium, kept on ice with the DNA, with a heat shock at degrees Celsius.
Step 2. (ii) Microinjection into the nucleus.
Step 3. (iii) The gene gun, with gold or tungsten particles coated with DNA.
Step 4. (iv) A disarmed pathogen vector, the changed Agrobacterium tumefaciens with a Ti plasmid.
Step 5. (v) A disarmed retrovirus, a changed virus used as a carrier.
Step 6. The insert breaks ampR, so the recombinant cells lose resistance to ampicillin. They still have tetR. They grow on tetracycline and not on ampicillin.
Answer: (i) heat shock; (ii) microinjection; (iii) gene gun; (iv) a disarmed Agrobacterium; (v) a disarmed retrovirus. The recombinants grow on tetracycline only.
Watch out. This is the reverse of the BamHI case, where the recombinants grow on ampicillin only.
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