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From 2010.igem.org

7/19

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.

7/21

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.

7/22

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.

7/23

Results: Inductinon 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.

7/27

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.

7/28

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

7/29

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.

8/11

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