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Team:Evry/Experimentals Parameters
From 2012.igem.org
Protocols to determinate the following experimentals parameters
Identify the auxin distribution in tadpole
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.
How to determine auxin diffusion from tadpole's skin 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. 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 ).
How to determinate the Auxin diffusion from Blood to Muscle
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 ). Then euthanize the tadpole and collect a volume of muscle. Determining the concentration of auxin in the piece of muscle removed. Repeat the experiment with at least 10 others tadpoles increasing the recuperation time of 5 minutes by experiments. Remark, you have to isolated always the same height of muscle. Thus, the values of auxin diffused from the blood to the muscle function of time will determine the desired parameters.
How to determinate the Auxin Half Life in the Skin
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 ). Then euthanize the tadpole and collect a volume of muscle. Determining the concentration of auxin in the piece of muscle removed. Repeat the experiment with at least 10 others tadpoles increasing the recuperation time of 5 minutes by experiments. Remark, you have to isolated always the same height of muscle. Thus, the values of auxin diffused from the blood to the muscle function of time will determine the desired parameters.
How to determinate the Auxin Half Life in the Muscle
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) .
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).
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.
Wait 5 minutes, and determine the auxin present in the tissue and the environment.
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
Repeat this experiment with at least 10 tadpoles, and by increasing the incubation time in the bath small volume of 5 minutes each time.
Finally just assign the value of auxin degradation over time to determine the parameters
How to determinate the Auxin Half Life in the Blood
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.
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.
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.
