Team:Evry/Experimentals Parameters

From 2012.igem.org

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<center><h1>Protocols to determine the following experimental parameters</h1></center>
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<center><h1>Protocols to determinate the following experimentals parameters</h1></center>
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<center><h2>Identify the auxin distribution in tadpole</h2></center>
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<h2>Auxin distribution in tadpole</h2>
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<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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<p> To determine where the auxin gather in tadpole, you have to inject the auxin directly in it's heart. In this way it will be distributed with the bloodstream. The next stop is tadpole quenching and detection of the auxin distribution by fluorescent anti-auxin antibody, which are comercially available. </p>
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<center><h2>How to determine auxin diffusion from tadpole's skin to the medium</h2></center>
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<h2>Determination of auxin diffusing from tadpoles to the medium</h2>
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<p> 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. After that tissue is transferred to MMR solution without auxine, and increase of auxin concentration in MMR is observed. It is thus possible to plot a curve (auxin quantity in fonction of time ). </p>
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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. 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 quantity as a function of time). </p>
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<h2>Determination of auxin diffusion from blood to muscle</h2>
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<center><h2>How to determinate the Auxin diffusion from Blood to Muscle</h2></center>
 
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<p> To determine the diffusion coefficient of auxin, it will have injected directly into the heart auxin. Then let the tadpole in the MMR for 5 minutes ( recuperation time ).
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<p> To determine the coefficient of auxin diffusion from blood to muscle, intracardiac auxin injection is done. After 5min of tadpole recuperation  in MMR, the tadpole is then  euthanized, and muscles are extracted to identify auxin concentrations in different samples.
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Then euthanize the tadpole and collect a volume of muscle.
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Determining the concentration of auxin in the piece of muscle removed.
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Repeat the experiment with at least 10 others tadpoles increasing the recuperation time of 5 minutes by experiments.
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Remark, you have to isolated always the same height of muscle.
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Thus, the values ​​of auxin diffused from the blood to the muscle function of time will determine the desired parameters. </p>
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<h2>Determination of auxin half life in tissues (muscle and skin )</h2>
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<center><h2>How to determinate the Auxin Half Life in the Skin</h2></center>
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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/>
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<i> Auxin concentration estimation in tissue was done by employing the massive volume difference between tissues and the bath. </i><br/>
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Then place the tissues in a small volume of MMR with the same auxin concentration.<br/>
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The volume of solution has to be equal to the volume of tissue.<br/>
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Here we know how much auxin was present in the bath of a small volume.<br/><br/>
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<p> To determine the diffusion coefficient of auxin, it will have injected directly into the heart auxin. Then let the tadpole in the MMR for 5 minutes ( recuperation time ).
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After 5 minutes,  determine the auxin presence in tissue and external environment.<br/><br/>
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Then euthanize the tadpole and collect a volume of muscle.
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Determining the concentration of auxin in the piece of muscle removed.
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Repeat the experiment with at least 10 others tadpoles increasing the recuperation time of 5 minutes by experiments.
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Remark, you have to isolated always the same height of muscle.
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Thus, the values ​​of auxin diffused from the blood to the muscle function of time will determine the desired parameters. </p>
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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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Repeat this experiment for  at least 10 tadpoles, but with prolonged incubation time in the bath of small volume.<br/>
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<center><h2>How to determinate the Auxin Half Life in the Muscle</h2></center>
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<p>On the half-life time of auxin in tissues, we must first load the tissue of auxin by soaking in a large volume of solution MMR with auxin (volume 10,000 times greater than the volume of tissue) .<br/>
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<i>So we know how much auxin was present in the tissue when it was removed from the bath at high volume (because we have the volume of tissue, and the concentration in the tissue is the same as in the bath due to the strong difference between the volume of tissue and that of the bath).<i><br/><br/>
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Then place the tissus loaded in a small volume of MMR with the same auxin concentration.<br/>
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The volume of solution has to be equal to the volume tissue.<br/>
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Here we know how much auxin was present in the bath of small volume.<br/><br/>
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Wait 5 minutes, and determine the auxin present in the tissue and the environment.<br/><br/>
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Then simply subtract this value from the degradation of auxin in the MMR only to know the amount of auxin degraded by tissue volume in 5 minutes<br/><br/>
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Repeat this experiment with at least 10 tadpoles, and by increasing the incubation time in the bath small volume of 5 minutes each time.<br/><br/>
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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 determinate the Auxin Half Life in the Blood</h2></center>
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<h2>Determination of auxin half-life in blood</h2>
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<p>About the half-life time of auxin in blood, you just have to get a volume of blood, and mix that with an auxin known quantity. Wait 5 minutes and determinate the concentration of auxin.<br/><br/>
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<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/>
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Finally, deduct the initial quantity of auxin prior incubation from that measured after 5min incubation. Thus, auxin degraded in 5min is measured.<br/>
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Repeat the experiment ten times, increasing the incubation time to trace the curve of auxin degraded in blood as a function of time. </p>
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Subtract the initial quantity of auxin added from the quantity mesurate in blood after 5 minutes to get the auxin degraded in the blood during the 5 minutes.<br/><br/>
 
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Restart the experiment ten time, increasing the wait time by 5 minutes to each time to trace the curve of the auxin degraded in blood fonction of time. </p>
 
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Latest revision as of 19:14, 10 October 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 quantity as 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 of tadpole recuperation in MMR, the tadpole is then euthanized, 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 by employing the massive volume difference between tissues and the bath.
Then place the tissues in a small volume of MMR with the same auxin concentration.
The volume of solution has to be equal to the volume of tissue.
Here we know how much auxin was present in the bath of a small volume.

After 5 minutes, determine the auxin presence 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 at 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



Determination of 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 times, increasing the incubation time to trace the curve of auxin degraded in blood as a function of time.



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