Difference between revisions of "Team:Valencia UPV/Experiments"

This procedure is done with the Promega; kit (Dual-Luciferase Reporter Assay System). Using Agrobacterium tumefaciens as a vehicle to insert the desired devise, it is necessary insert it into a leaf of N.benthamiana by a direct injection. This method is known as Agroinfiltration. The next step is letting infiltrated leafs for two or three days depending on how the experiment is programmed.

After two days post infiltration, users can get leaf disks from N.benthamiana using a hole punch. It is recommended to take the maximum agroinfiltrated area avoiding plant nerves. Leaf discs are put in a specific plate depending on the light condition requirements. Different samples are taken during the next two days after discs were made and immediately they are put in liquid nitrogen and stored at -80ºC. The steps to follow are:

1. The Passive lysis buffer 1x is prepared. Each disk of leaf needs 200µl. The passive lysis buffer is stored at 5X so it must be diluted with distilled water. Place it on the ice besides the LUCII substrate and the STOP solution.
2. Cut off two little leaf disks of approximately 0.8 cm and put it into an Eppendorf tube. Immediately, freeze it with liquid nitrogen to avoid the deterioration of vegetal material. Grind the freeze sample using the metabolomics robot. Put the samples on ice.
3. Add 150µl of passive lysis buffer 1x to each Eppendorf tube.
4. Vortex gently and centrifuge 13200rpm during 15 minutes at 4ºC. While switch on the luminometer.
5. Dilute 2:3 the extracts on a new Eppendorf tube. Add 36µl of Passive lysis buffer 1x and 24µl of sample.
6. Take an optimal plate to use in the luminometer. Fill luminometer wells with 40 µl of LUCII which is stored at -20ºC.
7. 10 µl of sample is added in each well. Wait 10min. During this time turn on and configure the luminometer.
8. Measure luciferase activity.
9. Prepare 40 µl/well of Dual Glo 1x (STOP solution + substrate). The substrate is at 50x concentration and stored at –20ºC.
10. When the first luciferase measure is done, it is necessary to add 40 µl of Dual Glo into each well. Let it rest during 10 min.
11. Measure the Renilla activity.
12. Take the obtained information and analyze it.

Things to keep in mind for the next experiment: The luminometer (machine to measure the luminescence) has to be ready before adding the reagents to the samples because it needs 10min to be ready. Set the timer (10min) with the first sample of luciferase and add the reactant to the other samples as quick as possible.

To carry out transient expression experiments, the method described by Orzaez et al. was followed (Orzaez et al. 2009). First of all, Agrobacterium tumefaciens GV3101 cultures incubated at 28ºC overnight were centrifuged at 3000rpm for 15 minutes. Cultures were pelleted and resuspended in 5mL of agroinfiltration solution (MES 10mM, pH 5.6, MgCl2 10mM and acetosyringone 200mM diluted with dimethyilsulfoxide (DMSO)).

Once pellet was resuspended, cultures were protected from light and incubated at room temperature for 2h under stirring conditions. Afterwards, cultures Optical Density at 600nm (OD600) was measured with a spectrophotometer, and readjusted to the desired OD by diluting with agroinfiltration solution. If coinfiltration with different genetic constructions is needed, A.tumefaciens culture with the corresponding plasmids were equally mixed.

Agroinfiltration mix was inoculated in 4 weeks old N. benthamiana plants using needleless syringes through abaxial surface of the three youngest leaves of each plant. Plant growing conditions were 16 light hours at 24ºC and 8 dark hours photoperiod at 20ºC. Finally, leaves samples were taken 5 days post infiltration (d.p.i.).