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Team:WashU/Notebook/Yeast
From 2010.igem.org
Week of 6/21
6/24
plated Yeast Gal-YFP MATa, trp1, leu2::GAL1-YFP URA3/Kanr and Yeast YF4 MATa Prototroph on a Kan plate and a YPD plate
Started a liquid culture of Yeast YF4 MATa Prototroph in YPD
Week of 6/28
6/29
A practice transformation was preformed at the Cohen Lab with Ilaria. The OD600 was 0.651 which was low, but used in order to speed up the experiment. The PMKV005 loxKanlox Plasmid (McCusker 2005 Yeast, 21: 163-171) was used into transform wildtype yeast. The plates used were Kan positive plates created on 6/24. However it was later found that these plates might not correctly select for drug resistance. Two plates were made, one with the transformant and one with a control. The attached procedure was used and annotated during the experiment
7/1
• Examined the Sample and Control plates from the practice transformation on June 29th. Two colonies had grown on the Sample plate and none showed on the control plate. The low colony number could be due to a low OD600 (0.651) used in the transformation. • Plated the two colonies on a G418 plate in order to conduct the first round of purification
7/2
PRACTICE YEAST TRANSFORMATION Colonies grew on the first purification plate for both colony 1 and 2. Two colonies were plated from both transformed colony 1 and transformed colony 2 onto a second and final purification plate -- PLATES: YPD: all yeast grew His-: only 1&2 (WT) yeast grew, (4,6,11,12 did not grow) Kan:3,4,7 grew- 1&2 were selected against but some colonies were visible Ura-: only 1&2 (WT) yeast grew, (11 did not grow)
Amp: bacteria 8,9,10 grew (other amp plate WT control did not grow)
conclusion: all the resistance plates work in that they select against those w/o the resistance; however, there was some low level of growth on some of these plates where there was not supposed to be
Week of 7/12
7/12
streaked out wild type yeast strain FY4 onto a YPD plate and inoculated about 5 mL of YPD- incubate and shake Brian: The FY5 freezer stock was accidentally allowed to thaw and therefor a second freezer stock was started by growing an overnight culture inoculated from the original freezer stock. The freezer stock was made according to protocol from the cohen lab.
7/13
Plated FY4 yeast onto a YPD plate to grow up for a practice transformation in case the plate from the freezer stock does not grown since there were no visible growths on it. The FY4 was taken from a Ura- plate plated on 6/29 because it had the best colonies out of all the plates in the refrigerator. A freezer stock of FY4 was made according to protocol from an overnight culture of the FY4 strain since the original stock was destroyed when plating on 7/12.
7/14
Yeast colonies were observed on the FY4 plated on 7/12 from the FY4 freezer stock. This plate was brought to the Cohen lab along with liquid YPD-4 and they were used to inoculate a 5ml overnight culture.
