Team:Valencia/WetLab/YeastTeam/Protocols

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== '''Protocol used to make our yeasts produce light''' ==
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'''PROTOCOL OF CITOPLASMATIC Ca2+ INCREASEMENT MEASUREMENT IN ''S. cerevisiae'''''
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Modified from the original Denis and Cyert (2002) JCB 156; 29-34.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Material:
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*<span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> pEVP11[AEQ] plasmid: apoaequorina expression (Batiza et al.(1996) J.Biol.Chem. 271: 23357-62).
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*<span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Coelenterazine solution: Diluted coelenterazine until 590μM in satured N2 metanol. This compound is extremely photosensible and it's inhibited by O2. Keep at –20ºC.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Note: We bought Coelenterazine, Native (CLZn) 50 μg Ref. C-2230 de
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">SIGMA. We put N2 gas into metanol during 5 minutes, and we added inmediately 200μL to the 50μg of coelenterazine.
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*<span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Luminometer.
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*<span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Luminometer tubes and ELISA plaques.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''Procedure:'''
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''1.''' We recieved pEVP11[AEQ] aequorin transformed yeast from Joaquin Arinyo.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''2.''' We let growing up o/n in SD lacking Leu medium to maintain plasmid expression.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''3.''' After incubation, measure OD a 660nm y calculate the necessary volum to obtain in 250μL a final OD of 1,8. Put that volum into an eppendorf tube with a hole in its tap.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''4.''' Centrifugate 1 minute at 13000rpm.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''5.''' Discard the supernatant.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''6.''' Resuspend the pellet into 250μL of fresh medium with coelenterazine 2μM (aprox. 3,5μL of coelenterazinestock solution / μL de medio).
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''7.''' Incubate during 5,5 horas at ambient temperature, in agitation and keeping in the darkness.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''8.''' Centrifugate 1 minute at 13000rpm. Discard the supernatant and resuspend in SD lacking Leu fresh medium without coelenterazine (see the proper volum below *).
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''9.''' Wait 15 min (yeast luminiscence is increased due to a peak of Ca2+ is induced by the glucose (Nakajimashimada et al. (1991) PNAS 88; 6878-82).
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''10.''' Measure basal luminiscence during 15 minutes.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''11.''' Add the correct reactive volum to induce luminiscence.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">In the chase of alcaline induction:
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''8.''' *Add 170μL of medium.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''9.''' Add 30μL of KOH 100mM.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Other stress types:
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">NaCl: 30μL NaCl 5M (0,75M final).
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">CaCl2: 30μL CaCl2 1.33M (200mM final).
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">KCl: 30μL KCl 100mM.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Note: yeasts should be treated secuentialy and in the same way to obtain reproducible results.
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<div style="position:absolute; top:150px; left:580px; overflow:hidden;">

Latest revision as of 16:52, 18 October 2009














Protocol used to make our yeasts produce light


PROTOCOL OF CITOPLASMATIC Ca2+ INCREASEMENT MEASUREMENT IN S. cerevisiae


Modified from the original Denis and Cyert (2002) JCB 156; 29-34.

Material:

  • pEVP11[AEQ] plasmid: apoaequorina expression (Batiza et al.(1996) J.Biol.Chem. 271: 23357-62).
  • Coelenterazine solution: Diluted coelenterazine until 590μM in satured N2 metanol. This compound is extremely photosensible and it's inhibited by O2. Keep at –20ºC.

Note: We bought Coelenterazine, Native (CLZn) 50 μg Ref. C-2230 de SIGMA. We put N2 gas into metanol during 5 minutes, and we added inmediately 200μL to the 50μg of coelenterazine.

  • Luminometer.
  • Luminometer tubes and ELISA plaques.


Procedure:


1. We recieved pEVP11[AEQ] aequorin transformed yeast from Joaquin Arinyo.


2. We let growing up o/n in SD lacking Leu medium to maintain plasmid expression.


3. After incubation, measure OD a 660nm y calculate the necessary volum to obtain in 250μL a final OD of 1,8. Put that volum into an eppendorf tube with a hole in its tap.


4. Centrifugate 1 minute at 13000rpm.


5. Discard the supernatant.


6. Resuspend the pellet into 250μL of fresh medium with coelenterazine 2μM (aprox. 3,5μL of coelenterazinestock solution / μL de medio).


7. Incubate during 5,5 horas at ambient temperature, in agitation and keeping in the darkness.


8. Centrifugate 1 minute at 13000rpm. Discard the supernatant and resuspend in SD lacking Leu fresh medium without coelenterazine (see the proper volum below *).


9. Wait 15 min (yeast luminiscence is increased due to a peak of Ca2+ is induced by the glucose (Nakajimashimada et al. (1991) PNAS 88; 6878-82).


10. Measure basal luminiscence during 15 minutes.


11. Add the correct reactive volum to induce luminiscence.


In the chase of alcaline induction:


8. *Add 170μL of medium.


9. Add 30μL of KOH 100mM.

Other stress types:

NaCl: 30μL NaCl 5M (0,75M final).

CaCl2: 30μL CaCl2 1.33M (200mM final).

KCl: 30μL KCl 100mM.

Note: yeasts should be treated secuentialy and in the same way to obtain reproducible results.

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