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Team:Evry/Experimentals Parameters
From 2012.igem.org
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| - | <center><h1>Protocols to determine the following | + | <center><h1>Protocols to determine the following experimental parameters</h1></center> |
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| - | <p> To determine | + | <p> To determine the site of auxin accumulation in tadpoles, intracardiac injection of auxin has to be done to ensure systemic distribution via the bloodstream. The next step is tadpole quenching followed by quantification and localization of auxin using commercially available fluorescent anti-auxin antibody. </p> |
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| - | <center><h2>Determination of auxin | + | <center><h2>Determination of auxin diffusing from tadpoles to the medium</h2></center> |
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| - | To determine the coefficient of auxin diffusion from the skin to the medium (MMR), tadpole's skin is soaked in MMR+auxin medium for 20minutes. | + | To determine the coefficient of auxin diffusion from the skin to the medium (MMR), tadpole's skin is soaked in MMR+auxin medium for 20minutes. Next, the tissue is transferred to MMR auxin-free solution. Then , elevation of auxin level in MMR is measured. finally, auxin level curve is plotted as auxin quantityas a function of time ). </p> |
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| - | <p> To determine the coefficient of auxin diffusion from blood to muscle, | + | <p> To determine the coefficient of auxin diffusion from blood to muscle, intracardiac auxin injection is done .After , 5min tadpole recuperation in MMR, the tadpole is then sacrificed, and muscles are extracted to identify auxin concentrations in different samples. |
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| - | + | <p>To determine the tissue half-life time of auxin, tissues were soaked in great MMR medium volume to recuperate auxin by diffusion.<br/> | |
| - | <p | + | <i> Auxin concentration estimation in tissue was done employing the massive volume difference between tissues and the bath. </i><br/> |
| - | <i> | + | |
Then place the tissus loaded in a small volume of MMR with the same auxin concentration.<br/> | Then place the tissus loaded in a small volume of MMR with the same auxin concentration.<br/> | ||
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Here we know how much auxin was present in the bath of small volume.<br/><br/> | Here we know how much auxin was present in the bath of small volume.<br/><br/> | ||
| + | After 5 minutes, determine the auxin present in tissue and external environment.<br/><br/> | ||
| - | + | Then the amount of auxin degraded by tissue volume in 5 minutes is calculated by deducting this value from the degradation of auxin in the MMR.<br/><br/> | |
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| - | Then | + | |
| - | Repeat this experiment | + | Repeat this experiment for least 10 tadpoles, but with prolonged incubation time in the bath of small volume.<br/><br/> |
Finally just assign the value of auxin degradation over time to determine the parameters</p> | Finally just assign the value of auxin degradation over time to determine the parameters</p> | ||
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| - | <center><h2>How to determine | + | <center><h2>How to determine auxin Half-Life in Blood</h2></center> |
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| - | <p> | + | <p>Auxin half-life time in blood is determined by mixing a known amount of auxin with a defined volume of blood.Incubate for 5 minutes and determine auxin concentration.<br/><br/> |
| - | + | finally, deduct the initial quantity of auxin prior incubation from that measured after 5min incubation. Thus, auxin degraded in 5min is measured.<br/><br/> | |
| - | + | Repeat the experiment ten time, increasing the incubation time to trace the curve of auxin degraded in blood as a function of time. </p> | |
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Revision as of 02:39, 27 September 2012
Protocols to determine the following experimental parameters
Auxin distribution in tadpole
To determine the site of auxin accumulation in tadpoles, intracardiac injection of auxin has to be done to ensure systemic distribution via the bloodstream. The next step is tadpole quenching followed by quantification and localization of auxin using commercially available fluorescent anti-auxin antibody.
Determination of auxin diffusing from tadpoles to the medium
To determine the coefficient of auxin diffusion from the skin to the medium (MMR), tadpole's skin is soaked in MMR+auxin medium for 20minutes. Next, the tissue is transferred to MMR auxin-free solution. Then , elevation of auxin level in MMR is measured. finally, auxin level curve is plotted as auxin quantityas a function of time ).
Determination of auxin diffusion from blood to muscle
To determine the coefficient of auxin diffusion from blood to muscle, intracardiac auxin injection is done .After , 5min tadpole recuperation in MMR, the tadpole is then sacrificed, and muscles are extracted to identify auxin concentrations in different samples.
Determination of auxin half life in tissues (muscle and skin )
To determine the tissue half-life time of auxin, tissues were soaked in great MMR medium volume to recuperate auxin by diffusion.
Auxin concentration estimation in tissue was done employing the massive volume difference between tissues and the bath.
Then place the tissus loaded in a small volume of MMR with the same auxin concentration.
The volume of solution has to be equal to the volume tissue.
Here we know how much auxin was present in the bath of small volume.
After 5 minutes, determine the auxin present in tissue and external environment.
Then the amount of auxin degraded by tissue volume in 5 minutes is calculated by deducting this value from the degradation of auxin in the MMR.
Repeat this experiment for least 10 tadpoles, but with prolonged incubation time in the bath of small volume.
Finally just assign the value of auxin degradation over time to determine the parameters
How to determine auxin Half-Life in Blood
Auxin half-life time in blood is determined by mixing a known amount of auxin with a defined volume of blood.Incubate for 5 minutes and determine auxin concentration.
finally, deduct the initial quantity of auxin prior incubation from that measured after 5min incubation. Thus, auxin degraded in 5min is measured.
Repeat the experiment ten time, increasing the incubation time to trace the curve of auxin degraded in blood as a function of time.
