Raw Experimental Notes June 2016

6/22/2016 Magi, Patrik, Kristen, Johan

Completed the following steps (as per plan!)

• Prepare define medium plates and broth
  • For the plates: we mixed SD-URA+ glucose+Agar
  • For the broth: we mixed SD-URA+ glucose
• Pour plates
• Save broth in fridge (to be used later)
• Culture yeast in medium
  • Culture control yeast in defined medium overnight (12-72 hrs)
  • Culture mutant yeast BY4741 in YPD overnight (12-72 hrs)
  • Culture mutant yeast BY4742 in YPD overnight (12-72 hrs)

Small plates were used (because we are out of large size plates). The small plates have a total volume of 14mL. We poured approximately 5mL per plate

Note: Need to purchase Agar and large plates

• Step 1B. Pour plates

6/20/2016 Magi, Patrik, Kristen

Planning for Next Steps: Verification of Yeast (Streaking) and Transformation Objective: Verify the phenotype of two yeasts by growing them on various media

Streaking:

Day 1 Wednesday (starting at 6): 3-4 hrs

• Step 0. Make & Autoclave define (SD-URA) medium broth (2 hrs) (x2 for plates and broth)
• Step 1A. Culture control yeast in defined medium overnight (12-72 hrs)
• Step 1B. Pour plates

Day 2 Friday 5 pm

• Step 3. Measure optical density & normalize amount that we grow on each plate (1/2 hr): Need to get someone to help with that
• Step 4. Streak on different plates (1/2 hr)
• Step 5. Freeze remainder (1/2 hr)

Day 3 (Sun/Mon) after 1-2 days to take pictures of plates to double-check that yeast and plates work correctly

• Take pictures of all plates

Transformation: goal is to do this by Monday 6/27

• Figuring Out Protocol & Confirming Reagents

Day 1 (some Monday when Patrik can come in in the morning) Step 0. 5 Hour Growth in Specific Medium (may require autoclaving)

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

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)
• agar (non-LB) is added at 20 g/ml

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)