I have tried some chemical and electrical methods, but it doesn't work :(
Come on, if you want a proper answer, you have to ask a proper question! Transform which plasmid in which organism?? If you are talking E coli, 10 kb is not a problem at all, you migtht want to check the antibiotic used, though.
Come on, if you want a proper answer, you have to ask a proper question! Transform which plasmid in which organism?? If you are talking E coli, 10 kb is not a problem at all, you migtht want to check the antibiotic used, though.
No, you don't need special methods or special hosts, 10 kb is not that large. Check that you are not using the wrong antibiotic in the selection plates and/or ensure that it is being used at the right concentration. Also use a positive control (a smaller plasmid) just in case, and have someone knowledgeable check your plating technique.
also,
1. Make sure that your cells are 'competent cells' for heat-shock or electroporation transformation. In general, cells need a special treatment to be competent. You can make your own competent cells or purchase from commercial companies.
2. Make sure that your specific gene products are not toxic to the cells.
For example, in GatewayTM cloning system, the ccdB gene, which is a potent gyrase inhibitor and is toxic to E. coli. In this case, special bacterial strain are needed. Plasmids containing ccdB gene can be propagated in the E. coli strain DB3.1, which harbors a mutation in the gyrase gene (gyrA462) that confers resistance to the toxic affects of the CcdB protein (see attachment).
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