User:Nadiezda Fernandez-Oropeza/Notebook/Notebook/2010/11/09: Difference between revisions
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== | ==The Antifade system== | ||
* | * This entry contains the recipe and procedure of the antifade system. | ||
===Procedure=== | |||
The antifade system contains: | |||
Glucose Oxidase (GOD) | |||
Catylase (CAT) | |||
β-mercaptoethano (BME) | |||
D-Glucose (not added directly to the antifade, but added to the motility solution) | |||
First, prepare the following solutions: | |||
* 100x solution of GOD in PEM | |||
The concentration is 2000 µg/ml. This is a very small amount of chemical, so it is easier to prepare a 1000x solution first and then dilute to obtain the 100x solution. Store the solution in the -20 <sup>o</sup>C fridge. | |||
* 100x solution of CAT in PEM | |||
The concentration is 800 µg/ml. This is an even smaller amount of chemical, so it is also better to prepare the 1000x solution first and then dilute it to obtain the 100x solution. Also note that this chemical is highly static and it will be a difficult task to transport it from the wax paper to the vial. | |||
Store the solution in the -20<sup>o</sup> C fridge. | |||
* 2 M D-Glucose in PEM ('''PEM-Glu''') | |||
Prepare a total solution volume of 1 ml. The best way to prepare this is to add 0.36032 g of D-Glucose to a micro-centrifuge tube. Then, add small amounts of distilled water and vortex until a good amount of chemical has dissolved. Repeat these last two steps until you reach the desired total volume. | |||
Prepare 20 µl aliquots and store them in the -80<sup>o</sup> C fridge. | |||
===Recipe for Antifade=== | |||
* 12 µl of 100x GOD in PEM | |||
* 12 µl of 100x CAT in PEM | |||
* 6 µl of BME | |||
Vortex the solution and spin it down. Prepare 5 µl aliquots and store in the -20<sup>o</sup>C fridge. I read that the aliquots should last up to a week under these conditions. However, it is my experience so far that they last only up to 5 days. | |||
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The Antifade system
ProcedureThe antifade system contains: Glucose Oxidase (GOD) Catylase (CAT) β-mercaptoethano (BME) D-Glucose (not added directly to the antifade, but added to the motility solution) First, prepare the following solutions:
The concentration is 2000 µg/ml. This is a very small amount of chemical, so it is easier to prepare a 1000x solution first and then dilute to obtain the 100x solution. Store the solution in the -20 oC fridge.
The concentration is 800 µg/ml. This is an even smaller amount of chemical, so it is also better to prepare the 1000x solution first and then dilute it to obtain the 100x solution. Also note that this chemical is highly static and it will be a difficult task to transport it from the wax paper to the vial. Store the solution in the -20o C fridge.
Prepare a total solution volume of 1 ml. The best way to prepare this is to add 0.36032 g of D-Glucose to a micro-centrifuge tube. Then, add small amounts of distilled water and vortex until a good amount of chemical has dissolved. Repeat these last two steps until you reach the desired total volume. Prepare 20 µl aliquots and store them in the -80o C fridge. Recipe for Antifade
Vortex the solution and spin it down. Prepare 5 µl aliquots and store in the -20oC fridge. I read that the aliquots should last up to a week under these conditions. However, it is my experience so far that they last only up to 5 days.
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