Why do we get non-specific colonies on SD-Ura and similar dropout medium plates in DNA containing and No DNA samples after the yeast transformation (LiAc-PEG method)? If transformation is successful then one should see it under the microscope (GFP for example) but these non-specific colonies do not show that signal however they can grow happily under the selection pressure (URA containing medium e.g.).When I check non-specific colonies of the yeast (S.cerevisiae) under the microscope they show presence of some kind of structures inside the cell. Does anybody know what those structures are? How to avoid this non-specific colonies?
Hi there,
What is the recipient strain for the transformation and what is its phenotype towards ura3? Is it point mutation (ura3-1 for instance) or deletion/insertion mutation (ura3-52 for instance)? If the former, the ura+ clones observed are spontaneous revertants (no revertant should be observed from deletion/insertion mutants). I don't see anything abnormal from your picture : one can see nucleus and vacuole inside the cells...
Hi Dominique,
It is a haploid yeast strain(4741) without URA3. I have transformed centromeric plasmid containing GFP-tagged protein into the this strain with URA3 as a selection marker. After transformation I got non-specific colonies(No GFP signal) on No plasmid control as well as the plasmid containing samples. I am curious about the structures that are close to the vacuoles and these structures are absent in the GFP-positives transformants. Do you think the structure is nucleus? If yes then why are they not seen in GFP-positives transformants?
As your strain BY4741 is actually deleted for URA3, you shouldn't get any background. So the trivial reason of getting clones in no plasmid control is that your strain is not pure (ie contaminated with ura+ cellst). So what I suggest is to plate the recipient strain on rich media so as to get isolated clones and then replica plating onto selective media (complete media lacking uracil) and start a new transformation experiment from one clone growing on rich media but dead on complete media lacking uracil. An other method to chase away ura+ cells is to plate your strain on rich media containing 5FOA which will specifically kill ura+ cells(ie growing cells will be ura-).
One common reason can be contamination of URA+ cells in your culture, even if you have a few cells of them, it can give colonies on both DNA+ as well as No DNA control.
Also, it can be becoz of URA contamination in media, with very small traces of URA in media, as a result of cross contamination.
AS U KNOW, i was also getting these structures in case of both GFP and Non-GFP cells. Exact answer i dont know, why these structures appear in the cell. But i think it has to do something with the growth conditions and the way we process cells before taking images.
If there is URA in the medium then there should be a lawn on the plates. But what I observed was distinct colonies looks all similar in shape and colour.
Thank you Asif and Dominique. What you are suggesting that I have already tried but not exactly in the same way.I have tried growing the untransformed (Only WT) on the SD-URA plate.These WT cells do not grow at all on SD-URA plate. So the question of mixing with URA+ is not there. The same cells after the heat shock and transformation, grow in SD-URA medium with some structures (Unknown may be) present inside.
If the strain is pure and ura-,then it might that ura+ contamination arises from the solutions used for the transformation (culture media, water, LiAc, PEG, DNA carrier)...
You may want to check your media like YNB and -Ura whether they have the exact components they should have or not. One way is using another batch.
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