• Prepare yeast from freezer stocks:(AH)
5.Control YFP
6.Control CFP
7. Gal: CFP-YFP

  • Each in 2.5mL YPD and streaked on YPD plates.


  • Yeast grew in liquid YPD to a visible degree only for culture 6.
  • Single colonies grew for 3, 4 and 6. No growth seen for 5 and 7.
  • 200uL each of 4, 5, and 7 liquid cultures were plated onto warmed YPD plates and incubated at 30C o/n.


  • From streaked out freezer stocks, single colonies of:
5.Control YFP
6.Control CFP

  • Were innoculated in 2.5mL of YPD, shaken at 30C (11am)
  • Cultures were spun down, washed 1X and then resuspended in 3mL YPG+1% raffinose (galactose=2%). (1am)
  • Dilute cultures were also made by adding 30uL of resuspended culture to 3ul YPG.
  • Returned to shake at 30C until 9am when cultures were transported to Cohen Lab for imaging.


  • Results:
    • Induction was sucessful, as fluorescent colonies were observed in 3 and 4. (control still needed).
    • However not 100% of cells showed fluorescence, probably due to severe contamination of yeast, later found to be a result of contaminated YPD.


  • New liquid cultures were made from freezer stocks for samples 3, 4, 5, 6, and 7 in both YPD and YPG.
  • Cultures will be used for imaging tomorrow.


  • Began microscope training with Olga on the Deltavision microscope.
    • Microscope slides were made from the cultures made yesterday. No E. coli contamination was noticed.
    • Microscope had problems focusing on samples. Fluorescence seemed to pervade everything, maybe due to bleaching.
    • Perhaps changing media will help


  • Set up cultures in 3mL YPD of:
5.Control YFP
6.Control CFP
7. Gal: CFP-YFP

  • Let grow for 12 hours shaking @ 30C, then washed 3,4,7 with YPG (Washed 5,6 with YPD) and shake for 12 more hours.


  • Went to the cohen lab to learn how to use their microscope.
  • We worked with Kim and Priya and were unable to obtain good images, they are going to set up an appointment with a microscope technician so that he can show us how to use it.
  • The folowing notes were taken:
    • UseImage J or AxiovisionLE for image quatitation programs
    • Find a floresence to volume ratio to adjust for cell size
    • CFP bleachs easily on a timframe of about 10s, this is visible, YFP takes much longer to bleach
    • We should standardize the exposure time with each fluorophore but not necessarily between the fluorophores
    • Use the Multidimensional Acquisition for 3 quick measures of brightfield, YFP and CFP
    • CFP bleaches YFP so do YFP first