Drummond:Fluorescence: Difference between revisions
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*Cell preparation | *Cell preparation | ||
#day before imaging: prepare yeast precultures in 2 mL YPsugar (YPD, YPsuc/raf) | #day before imaging: prepare yeast precultures in 2 mL YPsugar (YPD, YPsuc/raf) | ||
#day of imaging (allow for 3 hour induction): measure optical density (OD) of preculture. | #day of imaging (allow for 3 hour induction): measure optical density (OD) of preculture.<BR>Dilution may be necessary (1:20 dilution of preculture:growth media; 50 μL preculture/950 μL YPsugar) | ||
#normalize OD of yeast culture to 0.4 | #normalize OD of yeast culture to 0.4<BR>target absorbance (0.4)/original absorbance = ratio<BR>4000 μL (4 mL) * (ratio) = X μL preculture in 4 mL solution | ||
<BR>target absorbance (0.4)/original absorbance = ratio<BR> | |||
4000 μL (4 mL) * (ratio) = X μL preculture in 4 mL solution | |||
#Induce cultures with 2% galactose (for 4 mL culture, use 444.4 mL 20% galactose) | #Induce cultures with 2% galactose (for 4 mL culture, use 444.4 mL 20% galactose) | ||
#Incubate induced cultures at 30°C for 3 hours | #Incubate induced cultures at 30°C for 3 hours | ||
Line 20: | Line 18: | ||
#Resuspend cells in 500 μL PBS | #Resuspend cells in 500 μL PBS | ||
*Imaging | *LSM510 Imaging | ||
#Pipet | #Pipet 15 μL of sample onto standard glass slide (slide preparation ahead of time will allow for the settling of yeast cells) | ||
#Cover with # 1 1/2 slide coverslip | #Cover with # 1 1/2 slide coverslip | ||
#543 nm laser > ON, Argon laser (in standby) > slowly increase laser power to 60% | #543 nm laser > ON, Argon laser (in standby) > once ready, slowly increase laser power to 60% | ||
#Config > Channel | #Config > Channel mode > multi-track > TFP/mCherry configuration | ||
# | #Set 458 nm excitation level at 5% and increase as necessary | ||
# | #Set 543 nm excitation level at 50% and increase as necessary | ||
#Locate and focus on yeast cells using bright field and | |||
#Adjust pinhole, gain, offset, etc. as necessary | |||
==Materials== | ==Materials== | ||
*yeast growth media (YPD or YPsuc/raf) | |||
*yeast colony | |||
*water | |||
*PBS | |||
*20% galactose | |||
==References== | ==References== | ||
<biblio> | <biblio> | ||
</biblio> | </biblio> | ||
</div> | </div> | ||
{{Drummond_Bottom}} | {{Drummond_Bottom}} |
Latest revision as of 07:45, 26 June 2008
Introduction
Goal: to prepare yeast cells with RFP/TFP fluorescence for imaging on the LSM510.
Protocol
- Cell preparation
- day before imaging: prepare yeast precultures in 2 mL YPsugar (YPD, YPsuc/raf)
- day of imaging (allow for 3 hour induction): measure optical density (OD) of preculture.
Dilution may be necessary (1:20 dilution of preculture:growth media; 50 μL preculture/950 μL YPsugar) - normalize OD of yeast culture to 0.4
target absorbance (0.4)/original absorbance = ratio
4000 μL (4 mL) * (ratio) = X μL preculture in 4 mL solution - Induce cultures with 2% galactose (for 4 mL culture, use 444.4 mL 20% galactose)
- Incubate induced cultures at 30°C for 3 hours
- Remove 1 mL culture to 1.5 mL centrifuge tube
- Spin samples for 12 seconds at highest speed
- Pour off supernatant
- Resuspend cells in 1 mL H2O
- Spin samples for 12 seconds at highest speed
- Resuspend cells in 500 μL PBS
- LSM510 Imaging
- Pipet 15 μL of sample onto standard glass slide (slide preparation ahead of time will allow for the settling of yeast cells)
- Cover with # 1 1/2 slide coverslip
- 543 nm laser > ON, Argon laser (in standby) > once ready, slowly increase laser power to 60%
- Config > Channel mode > multi-track > TFP/mCherry configuration
- Set 458 nm excitation level at 5% and increase as necessary
- Set 543 nm excitation level at 50% and increase as necessary
- Locate and focus on yeast cells using bright field and
- Adjust pinhole, gain, offset, etc. as necessary
Materials
- yeast growth media (YPD or YPsuc/raf)
- yeast colony
- water
- PBS
- 20% galactose