Difference between revisions of "Team:UChile Biotec/Experiments"
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<p>The last reactive used was the Hydrogen peroxide because its function as the initiator and it was poured after 2 hours in order to let the Aptazyme interact with the toxin in addition only in the wells needed the volumes were adjusted with nuclease-free water to reach the 100 uL for the reaction! | <p>The last reactive used was the Hydrogen peroxide because its function as the initiator and it was poured after 2 hours in order to let the Aptazyme interact with the toxin in addition only in the wells needed the volumes were adjusted with nuclease-free water to reach the 100 uL for the reaction! | ||
| − | The Hydrogen peroxide was poured one row at a time, to prevent possible errors associated to the reaction rate constant, as it only takes about 3 minutes to be | + | The Hydrogen peroxide was poured one row at a time, to prevent possible errors associated to the reaction rate constant, as it only takes about 3 minutes to be complete.</p> |
<p> | <p> | ||
To measure the absorbance we used a Infinite 200 PRO NanoQuant Plate Reader from Tecan Trading AG. We measured the changes in the absorbance levels for 20 cycles every 30 seconds. | To measure the absorbance we used a Infinite 200 PRO NanoQuant Plate Reader from Tecan Trading AG. We measured the changes in the absorbance levels for 20 cycles every 30 seconds. | ||
Revision as of 01:40, 2 November 2017
Experiments
Protocols!
STX in Buffer HEPES
A master mix for each aptazyme sample was prepared following these instructions:
At the end of this procedure we obtained 7 different master mixes for each one of our samples (this includes de control without DNA). After this, each of these master mixes was heated in a dry bath for 5 minutes at 90ºc and after that they were left cooling down at room temperature for 25 minutes. In the meantime every reactive was prepared in a way that the final concentrations of each reactive in the final solution 8the one placed in every plate well) were as following:
When the master mixes were ready, we poured in every plate well the following volumes:
The last reactive used was the Hydrogen peroxide because its function as the initiator and it was poured after 2 hours in order to let the Aptazyme interact with the toxin in addition only in the wells needed the volumes were adjusted with nuclease-free water to reach the 100 uL for the reaction! The Hydrogen peroxide was poured one row at a time, to prevent possible errors associated to the reaction rate constant, as it only takes about 3 minutes to be complete.
To measure the absorbance we used a Infinite 200 PRO NanoQuant Plate Reader from Tecan Trading AG. We measured the changes in the absorbance levels for 20 cycles every 30 seconds.
in Buffer HEPES and Buffer PBST
Two sets of master mixes were made for each sample analyzed; these master mixes were made as followed:
Once the 2 sets of master mixes were ready, they were heated at 90ºc for 5 minutes and cooled down at room temperature for 25 minutes, in the meantime each reactive was prepared the same way as seen in the previous protocol for bufffer HEPES
As the concentrations of STX were different for each set of palte wells, we made 3 different preparations:
B. STX
0.33 uM STX
0.45 uM STX
The absorbance was measured with an Infinite 200 PRO NanoQuant Plate Reader from Tecan Trading AG in a period of 570 seconds. Each measure was made every 30 seconds.
Thermocycler configuration
The configuration used for the Thermocycler (only when needed) was the following:
