I want to quickly insert a Kanamycine resistance cassette into a gene of Neisseria dentiae (which is naturally competent).
To do so, I am planning to amplify by PCR the region containing my gene of interest, then digest the PCR product with a restriction enzyme that cuts in the middle of it. Then, I want to ligate another PCR product (the resistance cassette with the same restriction site on both ends) in between these two fragments.
My question is, can I ligate there 3 fragments (5'gene-KanamycinR-3'gene) and then transform that directly into Neisseria, without needing a plasmidic vector ?
Thanks
Well you can only if you do a second PCR from the ligation mix that amplifies the entire construct (Since you will have multiple ligation products some of which could be your construct or your construct in circular form since there a chance for blunt end ligation as well)
Yes. The overlap extension PCR is the way to go.
Other options:
1. You can get your gene synthesized by Genscript.
2. It appears that you want to create mutation in the genome of Nesseria by adding an antibiotic resistnace gene.
If that is the case then you don't need to add the whole gene rather about 50bp gene specific sequence on both ends of kanamycin gene fragment.
This can be easily achived by adding 50bp gene specific overhangs on the primers specific to the Kanamycin gene.
Hi Martin,
I recommend what Sudeep has said. In addition to it, I would like to know, what is the purpose for the insertion of an antibiotic resistance marker within the expression sequence of your gene? Would it not affect the natural gene sequence? If you still want, to avoid the use of the resistance marker-contaning plasmids; and tedious PCRs, you can place an order for the required sequence from commercial gene synthesizing brands.
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