Hello all.
I have plasmid which contains spectinomycin resistance gene.
Is it possible to replace it with kanamycin resistance gene or any other antibiotics resistance gene?
Thank you for reading my question.
It could be feasible depending of the cloning possibility of your vector, simply changing one K7 for another is possible but you have to make sure that your cloning won't disrupt your plasmid or the gene construct. All depend if the restiction sites flanking your antibiotic K7 can be used for the other one. Some commercial plasmids like pCDNA3 offers multiple version with different antibiotic selection marker
Yes, possible. However, this depends on the presence of restriction sites that will not disrupt essential elements in your plasmids.
1. Identify unique restriction sites immediately at the start and end of the spectinomycin resistance cassette
2. amplify kanamycin resistance cassette flanked with those restriction sites.
3. digest and remove the spectinomycin resistance cassette from vector
4. ligate digested kanamycin resistance cassette with the digested vector
If you have not changed the restriction sites you don't need to worry about whether the kanamycin gene is in frame with the promoter. It should be a simple switch.
Yes it is possible.....firstly cut unwanted resistance gene from plasmid and insert another resistance gene (generated by proofreading PCR with primers) . Just make sure that upon plasmid digestion with endonucleases 1 and 2 you don't remove from your plasmid essential elements as well.
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