I have strain with ura3-52 mutation. It could growth on SD-Ura in my first attempt. So I thought there is contamination and I requested for new strain from our colleagues. I checked it again and the cell are growing on SD-Ura, although the growth is slower than positive control (SD-Leu media, this strain isnt auxotrophic for Leucine). is somebody faced with same problem?
Now I am wondering this effects my experiments, because I want to use it as a selection marker for my plamid introduction into yeast cell.
Hi there,
Are you 100% confident in your SD-ura? (Is it selective for any other ura- strain you have in hands? Has the provider actually checked the strain on his side?)
To check the strain you may also try to grow it on 5FOA : if it's ura-, it will grow and if it's not it will die.
Dear Dominique,
They checked it on SD-Ura and for them seems to be ok, it is not growing. Unfortunately, we don’t have FOA. I try to amplify it by PCR and send it for sequencing.
YPH500 strain also has ura3-52 mutation and in our lab it is not growing on SD-Ura (at least in my previous experiments).
Hi again,
So streak the strain and YPH500 on the same plate and see what happen...
So if YPH500 is not growing and the strain is :
either the strain is actually not ura- (but it's opposite to what your provider stated)
or there is definitely something wrong in your SD-ura media (Is the simple addition of uracil to the plate enough to allow the growth of YPH500? Which would prove it's not selective for an other auxotrophy present in YPH500 and absent in the strain)...
It is strength, because this strain couldn't growth on SD-Ura plate, but it grows in liquid SD-Ura.
I already sequenced PCR amplified fragment from the genome of this strain, supposed to have ura3-52 mutation, but the result od sequencing is similar to functional URA3. Did I do something wrong? does somebody know about the sequence of ura3-52? I have not found it.
ura3-52 consists in an Ty insertion so URA3 sequence should be interrupted. Sequence alignment between the mutant and the wt should reveal the presence of the insert.
another approach to test your plates is to use a strain with a ura3DELTA NULL deletion such as the popular BY427 x series of strains. These should provide a reliable negative control.
what do you mean with "it grows in liquid SD-ura media". what is the rise in OD per time unit?
I cheked how looks yph500 growing in sd-ura (liqid culture). its seems yph500 also can grow in liquid very slowly but none of them grow on the sd-ura plate. the sequencing result for both strain is similar. anyway at the moment i could get final product by means of plasmid transformation, the plasmid has pathway genes and ura as a selection marker.
this growth seems to be related to the mutation type. strain containing ura3DELTA NULL deletion couldn't grow.
thanks you all for your suggestions
- Badges
- Science topic
- Similar topics
- Geoscience
- Asia
- India