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 where the auxin concentrates</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 concentrates, you have to inject first the auxin in the heart of the tadpole, for it is directly associated into the bloodstream. Then, after waiting 20 minutes that auxin diffuses throughout the body, fixing the tadpole to permeabilize the tissue, then a revelation by fluorescent antibody anti-auxin, visualizes the areas where concentrated auxin. </p>
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<h2>Determination of auxin  diffusing from tadpoles to the medium</h2>
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<center><h2>How to determinate the Auxin diffusion from Skin to Water</h2></center>
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<p> To determine the diffusion coefficient of auxin from the skin to the environment (MMR), just in time to first soak for 20 minutes a tadpole skin volume, the volume has been previously measured in a solution MMR with auxin. Then transferring the tissue in a solution of MMR, and every 5 minutes taking a constant volume of media for each samples it is possible to determine the desired parameter. In fact, each samples shows the quantity of auxin diffused from the skin to the media, during a period. It is thus possible to plot a curve ( quantities fonction of time ), and determine the parameters about Auxin diffusion from Skin to Water. </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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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).<br/>
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Then place it in another volume of MMR with auxin concentration but the same volume of solution equal to the tissues.
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Here we know how much auxin was present in the bath of small volume.<br/>
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Wait 5 minutes, then remove the fabric and the environment.<br/>
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Determine the auxin present in the tissue and the environment.<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/>
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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/>
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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>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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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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