I transformed a plasmid into yeast and selected for transformants by growing them in a selective media (liquid, not agar plate). My experiment requires that I continually grow/ split these transformants in this selective media and take aliquots at various timepoints, pellet the yeast via centrifugation, extract the DNA and sequence the population (by sequencing the plasmid).
An issue that has arisen is that at the earlier timepoints, there is still a fair amount of plasmid DNA that is floating freely in the culture, which is purified along with the yeast when I extract the DNA and shows up when I sequence. Typically I take an 50ml aliquot, pellet it in a falcon, resuspend in dH2O to wash, pellet, and repeat before extracting.
Is there a way of degrading/ digesting these unwanted extracellular plasmids without rupturing the yeast? Would some type of nuclease work? Would precipitating/ chelating the DNA work or would that cause the DNA to be pelleted along with the yeast during centrifugation?
I will do my best.
You select the plasmid in a liquid media but still appear when you repick the transformants? There should not be any plasmid when you select, unless you say are the transformants of the plasmid. When you say you pellet the Yeast, do you mean you remove the supernatant? By centrifuging the yeast you take the DNA? You extract the plasmid and take notice of the traits, characters of the genomes? (Maybe yeast as in Saccharomises cerevisae? ). A few resuspensions on your last step will help.
Of course you can extract nuclei and plasmids, but I don't think is like saying high copy number or lower copy number as you would do in bacteria... (The transformation is in a plasmid?) I don't think either you could take OD and make the difference between DNA and yeast cells... A DNAase could help but you should get the maximum number of yeast cells, although the plasmid characteristics should be taken in mind;double strand, +/-, etc. You could consider precipitating the cells, and leave the DNA floating(do you refere to whole plasmids? ). IMAC precipitation, magnets or maybe sepharose/agarose binding (affinity). If you quelate the DNA with Mg+2 you could take them with you or leave the yeast cells in the supernatant, but I think you can then separarate them in the next steps, but it wouldn't be the protocol you are saying (by changing gs or rpm).
The procedure takes 50 mL? (Resistance to an antibiotic? )
Please come and ask if I did not explain myself properly.
Thank you
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