Briefly, a common way to prepare the plasmid would be as follows. I am skipping a lot of details and small steps so as to provide an overview of the
procedure. I am sure you will be able to find detailed tutorials/explanations online for each of the steps.
1. Isolate genomic DNA from the luminescent bacterium (culture bacteria, isolate DNA)
2. use PCR to make lots of copies of the luminescence gene
3. Ligate the PCR product into a plasmid that you can propagate in E coli
4. Transform E coli with the ligation mixture
5. Isolate E coli colonies that contain the right gene by restriction tests+electrophoresis (verify the size of the insert) and preferably sequencing
the insert (rule out mutations in sequence introduced by PCR).
6. Culture more of the E coli containing the correct plasmid (lets call it pUrsa1), and isolate enough plasmid DNA from it for the next steps
7. Cut the Luminescence gene from pUrsa1, and at the same time cut open a suitable target plasmid (suitable for expressing the protein in your
organism of interest) with the same set of restriction enzymes. The target plasmid is usually bought or obtained from a friendly colleague.
8. Isolate the opened target plasmid and the insert containing the luminescence gene by electrophoresis, cutting the bands from the gel and isolating
the DNA from the pieces of gel.
9. Ligate the insert into the target plasmid
10. Transform E coli with it
11. Select colonies containing the correct plasmid (verify by cutting it with restriction enzymes and electrophoresis). Unlike PCR, restriction and
ligation reactions only very rarely introduce mutations, so no need to sequence it again.
12. Again, scale up the culture of the E coli containing the right plasmid and isolate the plasmid DNA from it.
However, you can order a plasmid containing an insert of any random sequence from companies that makes synthetic plasmids. For the cost of those
(expect around Eur 300,-) you can't even order the primers, enzymes, etc, you need. Never mind your time. Also, the convenience of being able to
design your insert with any random sequence (restriction sites, immune tags, etc) and having it arrive in two weeks while you are free to do other
useful things can't be beat so I highly recommend going that route.
I have no experience transforming fish. Your posts suggests to me that you should study how to transfer and express the gene in your target organism
(ie the fish) a bit more.
[Edited on 8-3-2017 by phlogiston] |
