Hello all. I am repeatedly streaking one yeast strain (MATa MAL2-8C, SUC2) in 5-FOA plates aiming to disrupt the URA3 gene. After spreading thrice, the yeast is still able to grow in SD-URA plates. Am I doing something wrong? Do you guys have any tips?
It is my first time trying this widespread protocol to select URA3- cells. PS: I solubilized 5-FOA in DMSO, filtered and added to autoclaved medium (w/ YNB, SD w/aa, glucose 20%, agar 2%) to final concentration of 100mg/mL. Unfortunately I cannot try to disrupt URA3 by using CRISPR in the moment.
Thank you all.
Do you add uracil to your plates containing 5-FOA (it doesn't seem so from your message)?
Also, are you sure your FOA plates are working? Do you have a positive control that you can add to the plates to test them?
I prepare my FOA plates by adding 0.1% 5-FOA in powder to hot agar (60-70 C) and stir with a stirrer bar then pour the plates (but I always check that the plates really work before using them!)
In addition to Marco Geymonat comments, how are you doing the disruption? Is this a directed disruption of some sort or are you just trying to isolate spontaneous mutations (which is possible but will be quite rare).
- Badges
- Science topic
- Similar topics
- Engineering
- Materials Engineering
- Powders