"

From 2010.igem.org

2010/07/19

• Prepare yeast from freezer stocks:(AH)

3.Gal-YFP
4.Gal-CFP
5.Control YFP
6.Control CFP
7. Gal: CFP-YFP

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

2010/07/21

• 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.

2010/07/22

• From streaked out freezer stocks, single colonies of:

3.Gal-YFP
4.Gal-CFP
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.

2010/07/23

• 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.

2010/07/27

• 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.

2010/07/28

• 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

2010/07/29

• Set up cultures in 3mL YPD of:

3.Gal-YFP 
4.Gal-CFP
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.

2010/08/11

• 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