2. Ligation in pUPD2:

The ligations have a total volume of 10 µl. All the parts were mixed together in an eppendorf of 0.2ml. The eppendorf was put in the thermocycler with the programs GB or GG, the differences between them are number of cycles. Explain the cycles!

*The cells with the asterisk are the ones that are going to be written down and specified in the lab-book. The others cells are constant unless we indicate it specifically on the lab-book.

The agroinfiltration is a process that consist of introducing Agrobacterium tumefaciens into a leaf plant by is underside. Agrobacterium tumefaciens is a bacteria that causes the formation of tumours in some plant species like Nicotiana benthamiana, the one that we are working on. This bacteria carried the plasmid that have the DNA construction we want to test and as it infects the cell plants produce a transitory expression of our DNA piece.

So to start the process of the agroinfiltration first of all we have to grow Agrobacterium in liquid culture two days, then refresh two times this first culture taking 5µl of the previous culture. The refresh cultures are only one day incubating at 28ºC. After this starts the procedure:

1. Centrifuge the Agrobacteriumcultures 10min at 3000rpm.

2. While doing this prepare the agroinfiltration solution. It is made of 10ml of MES 10x (100mM, pH 5.6) + 1ml MgCl2 (1M) + 100µl of acetosyringone solution (200mM) it is composed by 9.8mg of acetosyringone dilute with 250µl of DMSO. Add water up to 100ml.

3. Eliminate the supernatant of the cultures and then add 5ml of the agroinfiltration solution. Resuspend the bacteria and let them grow in dark in the shaker.

4. Measure the OD (optical density). To do this the Agrobacteriumculture is diluted in proportion 1:10 so it is put 900 µl of agroinfiltration solution and 100 µl of bacteria culture and then measure in the spectofotometer. Depending on the OD obtained the culture will be diluted with a quantity that gets the ODs to 0.2 (when infiltrating viral system the OD has to be 0.1).

5. The dilute bacteria is put in eppendorfs and are ready to agroinfiltrate.

Tips to agroinfiltrate:

• Do the infiltration on the young leafs without rough surface.
• Put the syringe with the solution that has bacteria in the undarside and gently introduce the liquid making also a bit of presure with the finger in the adaxial surface of the leaf.
• Change the gloves and the syringe each time you change construction that wants to be agroinfiltrated.
• Take the plants out in baches to avoid that due to de hot ambient they close their pores.

Before start:

This procedure is done with the PrΩ kit (Dual-Luciferase Reporter Assay System).

First of all is needed to agroinfiltrate the plant and let them for 2 o three days depending on how the experiment is raised. Normally in this days the plants are in darkness because our pieces to tests activates with different wavelegth of ligths. After two days discs are made, trying to take the maximum agroinfiltrated area without any nerve. The discs are put in the specific plate depending on in which ligth condition they need. The samples are taken during one or two days after the discs were made and inmediately the are put in liquid nitrogen and the storage in the -80ºC fridge.

The steps to follow are:

1. The Passive lysis buffer 1x is prepared. It is used 200µl per disc of leaf. The passive lysis buffer is 5x so diluted them with destilled water, always manipulate in ice.

2. Grind the freeze sample with a machine that shake the eppendorfs that had to have previusly two little metal balls or with plastic maces.

3. Add to the eppendorf 150µl of passive lysis buffer 1x.

4. Mix it with the vortex avoiding that they melt. Do this step in cold the maximum time possible.

5. The samples are centrifuged in cold during 15min at 13200rpm.

6. A dilution 2:3 is made with the extract, to do that put in a new eppendorf 36µl of passive lyssis buffer 1x and 24µl of sample.

7. The opaque plate to use in the luminometer is taken. 40 µl of Luciferase is added in each well.

8. 10 µl of sample is added too. Wait 10min. During this time turn and configurate the luminometer.

9. The luciferase activity is mesured.

10. 40 µl/sample +1extra of Dual Glo (1x) was prepared . The sustrate is at 50x and it is at –20ºC, the buffer to dilute it is in the fridge.

11. After the first masure is done add to the wells 40 µl of Dual Glo, let it 10 min and measure the Renilla.

12. Take the data obtained and analyze it.

Things to keep in mind for the next experiment:

• The luminimeter (machine to measure the luminescence) has to be ready before start adding the reactants 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.

Muy lejos, más allá de las montañas de palabras, alejados de los países de las vocales y las consonantes, viven los textos simulados. Viven aislados en casas de letras, en la costa de la semántica, un gran océano de lenguas. Un riachuelo llamado Pons fluye por su pueblo y los abastece con las normas necesarias. Hablamos de un país paraisomático en el que a uno le caen pedazos de frases asadas en la boca. Ni siquiera los todopoderosos signos de puntuación dominan a los textos simulados; una vida, se puede decir, poco ortográfica.

We make protoplasts in to different ways. At first we make protoplasts with a normal Nicotiana leaf and then we try to transform the alive protoplasts. After the infiltration at vacuum we can not obtain protoplasts, we change the method. First we agroinfiltrate leafs with the desired construction, we let them 3 hours in dark and then make the protoplasts. The general steps for every preparation is:

1. Prepare the enzymatic solution. It has 5mL of Mannitol (0.8M) + 200 µl KCl (1M) + 400 µl MES (0.5M, pH 5.7) + 150ng cellulase + 40mg Macerozyme + 4.2mL H₂O. Total volume for one preparation. Put it 10min at 55ºC.

2. Take out the solution and let it cool down.

3. Add 100 µl CaCl₂ + 4 µl Beta-Mercaptoethanol + 100 µl BSA (10%).

4. Put the enzymatic solution in a petri dish and cut the leaf in very thin strips. Put quickly the cut leafs so they do not dry.

5. In darkness, do the vacuum for 30min to the petri dish with the enzymatic solution and the cut leaf. Let the leaf 3h in darkness, no agitation.

6. Swirl the plate gently. Using a 5ml pipette tip (cut off the tip first) take the liquid and filter them with a 35-75 µm nylon mesh into a 13 mL tube. To clean the mesh add before a little bit of W5 solution. Also put the leaf stripes first and then throw the enzymatic solution.

7. Add 5ml of W5 solution.

8. Centrifuge at 100xg for 1min without brake.

9. Eliminate the supernatant. Leave a small volume so that the protoplasts do not dry.

10. Add WI solution till reach the desired concentration (10⁷ protoplasts per gram).

11. Finally put into the plate wells 250 µl of W5 and 100 µl of protoplasts solution.

Solutions:

W5: NaCl (154mM) + CaCl₂ (125mM) + KCl (5mM) + MES (2mM) + 17.8ml H20. Total volume of 50ml.

WI: MES (4mM)(pH 5.7) + NaCl (154mM) + CaCl₂ (20mM).

Before doing the essay the protoplasts are in a plate in the light conditions needed.

1. Take the solution with protoplasts and put it in an Eppendorf.

2. Centrifugue at 100xg for 1min.

3. Eliminate the maximum supernatant letting the protoplasts.

4. Put into liquid nitrogen and then keep it in the -80ºC fridge.

Start the essay:

5. Add to the freeze sample 100µl of Passive lysis buffer (1x).

6. Vortex the samples.

7. Let it 5min in ice.

8. Centrifuge it at 1000xg for 2min.

9. Eliminate the supernatant.

10. Take an opaque plate to measure the luciferase and put in each well 40 µl of Luciferase and 10 µl of sample, wait 10min and then measure.

11. Add to the wells 40 µl of Dual Glo (1x) and measure the renilla luminiscence.