Here we present you all the procedures we did to develop our project. On this page you can find the general protocols. If preferred, you can go directly to the dialy Notebook, the experiments on Nicotiana or the protoplasts experiments by pressing in the buttons above or below (after protocols). We hope you enjoy reading our incredible journey!
Constructions protocol
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.
DNA; pUPD2
1 µl DNA fragment
1 µl pUPD2
1.2 µl buffer ligase
1.2 µl BSA (10x)
1 µl BsmbI
1 µl T4 ligase
5,6 µl H2O
8. Ligation in α or Ω:
DNA1;pUPD2+DNA2;pUPD2 ; α
DNA1; α1+DNA2; α2; Ω
1 µl DNA1; pUPD2
1 µl DNA1; α1
1 µl DNA2; pUPD2
1 µl DNA2; α2
1 µl α
1 µl Ω
1.2 µl buffer ligase
1.2 µl buffer ligase
1.2 µl BSA
1.2 µl BSA
1 µl T4 ligase
1 µl T4 ligase
1 µl BsaI
1 µl BsmbI
4.6 µl H2O
4.6 µl H2O
3a. Transformation:
In order to transform the DNA construction the electroporation method was used.
The method followed is common for E. coli and Agrobacterium. The electroporation cuvette was put in ice 10 minutes before inserting the cells.
Frozen cells were taken out of the -80ºC freezer, and they were put immediately into ice.
1-2 µl of the ligation were taken and added carefully to the electrocompetent cells.
60 µl of the mix were taken and put into an electroporation cuvette making sure that there were no bubbles.
The cuvette was dried and put in the electroporator, making sure that it did not do a spark. In that case, the process did not work and must be repeated.
The voltage is 1500V for E. coli and 1440V for Agrobacterium.
Then with 300 µl of medium the electroporated cells were taken and put into an Eppendorf, letting them grow in the shaker.
SOC medium was used for E.Coli and they were put at 37ºC for 1h.
LB medium was used for Agrobacterium and they were grown for 2h at 27ºC.
3b. Petri dish culture:
Depending on the plasmid with which the bacteria was transfected, agar dishes with the specific antibiotic were needed to make the petri dishes cultures.
E. coli-pUPD2 plasmids: chloramphenicol.
E. coli-Alpha 1 and 2: kanamycin.
E. coli-Omega 1 and 2: streptomycin
Agrobacterium: rifampicin + the specific one for each construction.
The procedure was made in the laminar flux cabinet. The spread plate method is done with 50-40 µl of the bacteria culture that is in the eppendorf. It was spread with the glass dipstick. After that the plates were put for 16h approximately in 37ºC for E. coli and 32h at 28ºC for Agrobacterium.
4. Liquid culture:
For Escherichia coli:
The mix was grown 16h at 37ºC in the shaker.
For Agrobacterium tumefaciens:
The mix was grown 32h at 28ºC in the shaker.
5. Minipreps:
In order to do the minipreps -extraction of the plasmids out of E. coli the protocol of the Omega kit (Plasmid DNA Mini Kit I Spin Protocol) was used. The steps to do it are:
1. Centrifuge at 10.000xg for 1minute at room temperature the liquid medium with the growed bacteria.
2. Decant or aspirate and discard the culture media.
3. Add 250 µl SolutionI/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining goo yields.
4. Tranfer suspension into a new 1.5mL microcentrifuge tube.
5. Add 250 µl Solutions II. Invert and gently rotate the tube several times to obtain a clear lysiate. A 2-3 minute incubation may be necessary.
6. Add 350 µl Solution III. Inmediately invert several times until a flocculent white precipitate forms.
7. Centrifuge at maximum speed (>13.000xg) for 10 minutes. Acompact white pellet will form. Promptly preceed to the next step.
8. Insert a HiBind DNA Mini Column into a 2 mL Collection tube.
9. Transfer the cleared supernatant from Step 8 CAREFULLY aspirating it into the HiBind DNA Mini Column. Be careful not to disturb the pellet and that mo cellular debris is transferred the the HiBind DNA Mini Column.
10. Centrifuge at maximum speed for 1 minute.
11. Discard the filtrate and reuse the collection tube.
12. Add 500 µl HBC Buffer.
13. Centrifuge at maximum speed for 1 minute.
14. Discard the filtrate and reuse collection tube.
15. Add 700 µl DNA Wash Buffer .
16. Centrifuge at maximum speed for 1 minute.
17. Discard the filtrate and reuse the collection tube.
18. Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column.
19. Transfer the HiBind DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
20. Add 30-100 µl Elution Buffer or sterile deionized water directly to the center of the column membrane.
21. Let sit at room temperature for 1 minute.
22. Centrifuge at maximum speed fot 1 minute.
6a. Digestion:
After doing the miniprep the DNA was obtained. The next components were mixed up in a 200 µl eppendorf. After the mix was done it stayed at 37ºC, 1h.
1 µl of the DNA
1 µl specific buffer
0.5 µl of the specific enzyme
7.5 µl of H2O
1 µl of loading buffer is needed for each 5 µl of the digestion mix, so they were added 2 µl of loadding buffer (6x).
These are the specific enzymes and buffers for each type of plasmid.
Plasmid
Enzyme
Buffer
pUPD2
Not I
Orange
Alpha
EcoRI
Specific
Omega
BamHI
Specific
The plasmids can also be cut with other enzymes if it is necessary to check the construction.
6b. Gel:
The gel was made with buffer + dilution 1:1000 of ethidium bromide and a proportion of 0.1% of agarose.
The small gels had 40ml of buffer + 0.4 µl of ethidium bromide and 0.4g of agarose.
After waiting 1h to let the gel cool down, the ladders of 100bp and 1kbp are put one on each side of the gel, and the digestions in between the ladders.
The voltage to apply is 120V.
It was written in a table the DNA fragments obtained and the words “ok “ or “no” depending on if the results are correct or not. Example:
DNA1
DNA2 C1
DNA2 C2
DNA4
ok
no
no
ok
7. Sequence:
To check if the plasmid obtained has the proper construction, a Sanger sequencing was made.
The IMBCP has its own sequencing service.
Agroinfiltration protocol
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.
Luciferase assay protocol
Before start:
This procedure is done with the Promega; 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.
Western blot protocol
DAY 1
PROTEIN EXTRACTION
1. Harvest agroinfiltrated leaves. Grind the harvested material in liquid N2. You can store ground tissue at -80ºC.
2. Weigh around 100 mg of ground tissue in a 1.5 ml tube. Keep sample frozen!
3. Add 3 vol. ice-cold extraction buffer to each sample (300 µl buffer/100 mg tissue).
4. Mix thoroughly by vortexing for a few seconds. Transfer samples to ice. Repeat vortexing a few times (cooling the sample in between) till sample is completely thaw.
5. Centrifuge the extract for 15’, at > 12000xg, 4ºC.
6. Transfer the supernatant to a fresh 1.5 ml tube. Samples should be kept cold at all times, work on ice!
SDS-PAGE
1. Sample Mix Preparation ( for 10 µl final volume, scale up as necessary):
Protein extract
1 to 6.5 µl
NuPAGE LDS Sample buffer (x4)
2.5 µl
NuPAGE LDS reducing agent (x10)
1 µl (use only for reducing conditions)
Ultrapure H2O
0 to 5.5 µl
Mix by vortexing. Heat at 72ºC(*) for 10’. Spin briefly to collect everything at the bottom of the tube. Keep samples on ice.
2. Assembling the gel and loading the samples:
Prepare 800 ml MES SDS 1x running buffer
20x MES MES SDS running buffer
40 ml
Distilled H2O
760 ml
Set aside 200 ml of 1x running buffer. Add 500 µl of NuPAGE antioxidant (only for reducing conditions). Mix by inversion.
Take one 10% Bis-Tris NuPAGE gel out of the plastic bag and rinse with distilled H2O. Peel off the white tape at the bottom of the gel.
Pull out the comb.
Insert the gel in the sure lock gel. The shorter well side of the cassettes facing inwards. Lock the gel tension wedge.
Fill the upper buffer chamber with the 200 ml of 1x running buffer with antioxidant. Fill the lower buffer chamber with remaining 600 ml of 1x running buffer.
To load the samples: insert the tip into the well and slowly pipet the sample into it.
3. Running the gel:
Running conditions: 200 V, 40 min
PROTEIN TRANSFER
1. Preparing for transfer:
Cut 1 piece of Hybond-P PVDF membrane and 2 pieces of whatman paper of the same size of the gel (8 x 7 cm).
Prepare 500 ml of Transfer buffer:
Transfer buffer (x20)
25 ml
Methanol
50 ml
H2O
375 ml
Soak 5 blotting pads in transfer buffer. Remove air bubbles by squeezing the blotting pads while they are submerged in buffer (this step is important because air bubbles may block the transfer of proteins).
Pre-wet the PVDF membrane for 10’’ in methanol. Wash in distilled water for 5’. Equilibrate in transfer buffer for at least 10’ before blotting. (Everything is done without agitation).
Wathman paper: soak briefly in transfer buffer immediately before using.
Lay the gel on the bench allowing one edge to hang 1 cm over the side. Insert the gel-knife into the gap between the gel plates and push up and down gently to break the bonds that hold the plates together. When all the bonds are broken separate the two plates and throw away the one without the gel.
Cut the wells with the gel-knife.
Place a piece of pre-soaked whatman paper on top of the gel. Keep the filter paper saturated with transfer buffer and remove all trapped air bubbles by gently rolling a glass pipette over the surface.
Turn the plate over so that the gel and whatman paper are facing downwards over your hand or over a piece of parafilm on the bench.
Remove the gel from the plate: use the gel knife to carefully loosen the bottom of the gel so that it peels away from the plate.
3. Transferring the gel:
Wet the surface of the gel with transfer buffer and place a pre-soaked membrane on top of the gel. Make a small cut on one corner of the membrane to mark the orientation. Remove air bubbles by rolling a glass pipette over the membrane surface.
Place a pre-soaked whatman paper on top of the membrane. Remove air bubbles.
Place 2 soaked blotting pads into the Xcell II Blot Module. Place the paper-gel-membrane-paper sandwich on top of the blotting pads.
Add another 3 pre-soaked blotting pad on top of the sandwich.
Place the blot module in the buffer chamber. Lock the gel tension wedge into place.
Fill the blot module with transfer buffer until the gel-membrane sandwich is covered in buffer (do not fill all the way to the top as this generates extra conductivity and heat).
Fill the outer buffer chamber with ˜ 650 ml deionized water.
Running conditions: 30V for 2h
BLOCKING THE MEMBRANE
1. Prepare 50 ml of blocking solution.
2% ECL Advance Blocking Agent in PBS-T (pH 7.5):
1 g blocking agent
50 ml PBS (1x)
50 µl Tween-20
2. Take the membrane out of the blotting module and transfer it to the blocking solution (protein side up, work always this way from now on). Leave o/n at 4ºC.
DAY 2
DETECTION
1. Prepare 2 L of wash buffer (PBS-0.1% Tween (pH 7.5))
200 ml PBS 5x (pH 7.5)
800 ml distilled H2O
1 ml Tween 20
3. Prepare 10 ml of antibody diluent (2% ECL Advance blocking agent in PBS-0.1%Tween) for each antibody you are going to use.
4. For 6x His-tag detection: dilute the Anti-His6 mouse monoclonal antibody 1:2000 in 10 ml of antibody diluent (5 µl antibody in 10 ml antibody diluent). Incubate for 1h at RT on a shaker.
For IgA detection: dilute the Anti-IgaH antibody 1:20000 in 10 ml of antibody diluent (0.5 µl antibody in 10 ml antibody diluent). Incubate for 1h at RT on a shaker. This antibody is already conjugated to HRP and does not need a secondary antibody for detection; go directly to step 8 after the 1 incubation.
5. Discard the antibody solution and wash the membrane with wash buffer:
2 x brief wash
1 x 15’ wash (RT, shaker)
3 x 5’ wash (RT, shaker)
7. Incubate the membrane with the secondary antibody for 1h at RT on a shaker.
8. Repeat step 5.
9. Take the detection reagents from the fridge and allow to equilibrate to RT before opening.
10. Mix detection solutions ECL Plus A and B in a ratio 40:1.
975 µl Sol. A + 25 µl Sol. B (enough for 1 membrane)
11. Drain the excess wash buffer from the membrane by holding the membrane gently with forceps and touching the edge against a tissue. Place the membrane protein side up on an acetate sheet. Pipette the mixed detection reagent on to the membrane.
12. Cover the membrane with another acetate sheet and gently smooth out any air bubbles, but do not apply pressure over the membrane. Dry any extra liquid with tissue.
13. Place a piece of filter paper on a X-ray film cassette (fix with tape). Drain excess of detection solution with a tissue and place the wrapped blots on top of the paper (fix with tape). Close the cassette and take to the dark room together with the autoradiography films and timer.
14. Switch on the film processor (front panel, lower right corner). Check that the temperate selector on top of the switch on button is on position 2. Turn off the light and with the red light on open the cassette and place a sheet of autoradiography film on top of the membrane (bend the lower right corner to mark the orientation). Close the cassette and expose the film for 1’. To develop the film place it on the rear feeding tray (shorter side of the film opposite to the bent corner against the feeding area to minimize the possibility that the film gets stack in the processor) and press the button next to it to start the feeding. Adjust exposure time as necessary.
Protoplasts protocol
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.
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).
Protoplast luciferase assay protocol
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.
