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Team:WashU/Notebook/Microscropy
From 2010.igem.org
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*200uL each of 4, 5, and 7 liquid cultures were plated onto warmed YPD plates and incubated at 30C o/n. | *200uL each of 4, 5, and 7 liquid cultures were plated onto warmed YPD plates and incubated at 30C o/n. | ||
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*From streaked out freezer stocks, single colonies of: | *From streaked out freezer stocks, single colonies of: | ||
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*Returned to shake at 30C until 9am when cultures were transported to Cohen Lab for imaging. | *Returned to shake at 30C until 9am when cultures were transported to Cohen Lab for imaging. | ||
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==2010/07/23== | ==2010/07/23== | ||
*Results: | *Results: | ||
Latest revision as of 07:18, 27 October 2010
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
