Im using pkD46 transformed competent cells and transforming with DNA template containing kanamycin resistance gene amplified.
I used pkD4 for amplifying the Kanamycin gene.
During my transformation experiments, Im getting a few false positives which are the cells that are transformed with reminisces of pkD4 plasmid. This is happening even after Dpn1 digestion of the template to remove the plasmid background.
What can be done to remove the background completely?
Have you confirmed they contain the pKD4 plasmid and that they're not just spontaneous kanamycin resistant mutants? Those spring up sometimes, to greater or lesser extent depending on the species and strain you're working with. In that case, my only advice would be to double the kanamycin concentration or just screen more colonies.
Also, pKD4 has a R6K suicide origin so it shouldn't replicate at all unless your strain has an R6K plasmid relative, or you're working with a strain that has the pir gene integrated into the chromosome. If they are actually transforming with pKD4 then my advice would be to just get a good gel extraction kit and gel extract your PCR fragment. You can always re-amplify from gel purified template if the concentration is too low to use.
Hi there,
If your false positives actually contains pKD4 plasmid, you might get rid of them by either gel purifying the cassette prior to transformation or linearize the plasmid by restriction endonuclease digest (which will prevent cell transformation with circular plasmid. No site should be present within the cassette)
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