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Team:Valencia/WetLab/YeastTeam/Protocols
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| - | Modified from the original Denis and Cyert (2002) JCB 156; 29-34. | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Modified from the original Denis and Cyert (2002) JCB 156; 29-34. |
| - | Material: | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Material: |
| - | - pEVP11[AEQ] plasmid: apoaequorina expression (Batiza et al. | + | *<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). |
| - | (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. | + | *<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. |
| - | Note: We bought Coelenterazine, Native (CLZn) 50 μg Ref. C-2230 de | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">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. | + | <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. |
| - | - Luminometer. | + | *<span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Luminometer. |
| - | - Luminometer tubes and ELISA plaques. | + | *<span style="color:black; align:justify; font-size:11pt; font-family: Verdana"> Luminometer tubes and ELISA plaques. |
| - | '''Procedure:''' | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''Procedure:''' |
<div align="justify" style="position:relative; top:-5px; left:70px; width:500px"> | <div align="justify" style="position:relative; top:-5px; left:70px; width:500px"> | ||
| - | '''1.''' We recieved pEVP11[AEQ] aequorin transformed yeast from Joaquin Arinyo. | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''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. | + | <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. |
| - | '''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. | + | <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. |
| - | '''4.''' Centrifugate 1 minute at 13000rpm. | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''4.''' Centrifugate 1 minute at 13000rpm. |
| - | '''5.''' Discard the supernatant. | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''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). | + | <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). |
| - | '''7.''' Incubate during 5,5 horas at ambient temperature, in agitation and keeping in the darkness. | + | <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. |
| - | '''8.''' Centrifugate 1 minute at 13000rpm. Discard the supernatant and resuspend in SD lacking Leu fresh medium without coelenterazine (see the proper volum below *). | + | <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 *). |
| - | '''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). | + | <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). |
| - | '''10.''' Measure basal luminiscence during 15 minutes. | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''10.''' Measure basal luminiscence during 15 minutes. |
| - | '''11.''' Add the correct reactive volum to induce luminiscence. | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''11.''' Add the correct reactive volum to induce luminiscence. |
| - | In the chase of alcaline induction: | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">In the chase of alcaline induction: |
| - | '''8.''' *Add 170μL of medium. | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''8.''' *Add 170μL of medium. |
| - | '''9.''' Add 30μL of KOH 100mM. | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''9.''' Add 30μL of KOH 100mM. |
| - | < | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">Other stress types: |
| - | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">NaCl: 30μL NaCl 5M (0,75M final). | |
| - | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">CaCl2: 30μL CaCl2 1.33M (200mM final). | |
| - | + | <span style="color:black; align:justify; font-size:11pt; font-family: Verdana">KCl: 30μL KCl 100mM. | |
| - | + | <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. | |
| - | + | ||
| - | Note: yeasts should be treated secuentialy and in the same way to obtain reproducible results. | + | |
<div style="position:absolute; top:150px; left:580px; overflow:hidden;"> | <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.
