Experiment to determine whether yeast is contaminated and to salvage strain using 5FOA, 23Oct2014
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October 23, 2014: Experiment to determine whether yeast is contaminated and to salvage URA- strain from possibly mixed sample
- Location: BioCurious
- Participants: Lafia, Johan
- Notes: Lafia, Johan
- Aims: Our yeast strains may be contaminated, so Rachel designed an experiment that would help us determine what strain we have and whether they are mixed. If indeed we have mixed or URA- strains, this experiment would allow us to pick out the URA- colonies so that we may use it. It would also give us confidence that we have a viable selection media to use when we do yeast transformations.
- 12 plates from a new sleeve acquired at Biocurious
- 8 plates (4 each of YPD and YPD w/URA) pre-made by Teknova from a new sleeve from the batch ordered by Craig
- 250 ml YPD Agar + 5FOA, Prepared using Teknova YPD Broth from batch ordered by Craig. Agar was also from Teknova, ordered by Johan. 2.5 ml of 100x liquid 5-FOA was added after the media cooled below 45 degrees.
- 250 ml CM minus URA powder media ordered by Patrik (5FOA added after some of the media was used to make plates)
- Media prep:
- 250 ml of Teknova broth and 5g of Agar was autoclaved in a glass bottle. Upon cooling below 45 degrees (as measured with an Extech IR thermometer), 2.5 ml 5FOA was added.
- 250 ml of CM minus URACIL liquid media was prepared in a glass bottle using 8g of powder and 4g Agar.
- Plates streaking:
- 4 plates were prepared of each of the 5 media types (20 total). The media types were YPD, YPD+URA, YPD+5FOA, CM, CM+5FOA. For each media type, the plates were streaked with A) Shashank's yeast strain acquired from Carolina, B) Counter Culture Labs' yeast strain from Carolina, C) LA Biohacker's yeast strain marked BY-4741, D) blank loop (control). The samples were streaked in a manner that should produce single colonies. Sterile plastic loops from a new bundle were used. A new loop was used for each streak direction. During streaking, aseptic technique was used: All work was done within the hood. The lip of the sample bottles were flamed each time prior to opening. The hood surface was wiped down with 70% alcohol and was subject to 30 minutes UV sterilization.
- Media prep: