Raw Experimental Notes June 2016

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6/13/2016 - Magi, Kristen, Patrik, Sean

Experiment: Yeast Revival & Confirmation of Correct Yeast

Tasks:

  • 0. Revive yeast cultures (Patrik did this previously, and confirmed that the baker's yeast (control) was able to produce its own Uracil; however, the revived cell did not appear to exhibit much growth, so additional 500l of yeast strain was added to the tube, and another tube was also made) - Complete
  • 1. Prepare agar mixes (done) - Complete
  • 2. Pour Plates - Complete
  • 3. Streak plates & then check for colony growth - Pending

ToDo(s):

  • Immediate: Confirm that the yeast cultures exhibit the correct behavior (ability to grow in 5-FOA and not in SD-URA)
  • Next Steps: Make new glycerol stock
  • Later (Next Week): transformation (assuming everything goes well)


  • Found previous prepared plates (large & labeled RVC)
  • Check for contamination: 4 -URA + Glucose (colorless), 5 (mixture of YPD + YPD + FOA):> decided not to use these and to repaired
  • Pouring medias: YPD, SD (-Ura -Glu), 5-FOA; decided to pour whole pack (25), so 8 plates of each
  • Volume of plate: 700 ml per plate; decide to make 20 ml of working media (so 20 x 8 plates = 160 ml)
  • YPD = 50 g/L; SD- = 7.47 g/L; (need to add 20 g/L of glucose); 5-FOA: 0.5% see prep calculations here: https://docs.google.com/spreadsheets/d/1SRrFP4an20c3uaTYZRlUEeXM6md2O3ES7In1e4Jjqnk/edit#gid=0
  • Last night Patrik started some cultures: 1) control 2) revived culture (from the glycerol stock): should be able to grow in presence of 5-FOA but not in SD-URA
  • Autoclave settings: 121 C (setting at 21)


Practices:

  • Plates should be stored upside to avoid collecting contamination on lids)
  • Contaminated plate submerged in bleach water
  • Preparing bleach: 1:10 bleach to tapwater (grab from restroom; not hand-washing water)

06/15/16 Patrik & Magi

Several of the standard URA- yeast strains grow poorly in defined medium, because the have a leucine uptake defect. So we decided to pour some plates with defined medium and double the normal amount of leucine. Standard leucine concentration in SD is 120mg/L.

See:

Commonly used Saccharomyces cerevisiae strains (e.g. BY4741, W303) are growth sensitive on synthetic complete medium due to poor leucine uptake
http://onlinelibrary.wiley.com/enhanced/doi/10.1111/j.1574-6968.2007.00798.x/

Ingredients:

  • 7.47g/L SD-URA-Glucose
  • 20g/L glucose
  • 20g/L agar
  • 120mg/L leucine

Adding leucine at 120mg/L in a 160mL mix which equates to 19.2mg of Leucine- used 23.4mg (accuracy achieved on scale)

In 160mL we added: 23.4mg of Leucine, 3.2g of Agar, 1.15g of SD-Ura-Glucose and 3.2g of Glucose

Added to autoclave until it reaches 121C then removed by Patrik.

Next steps:

  • Get ready for Electroporation:
    • Figure out the ideal timing to perform electroporation- ideally during the log phase of the yeast growth phase
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