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− | + | .page-header{ | |
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− | + | } | |
+ | @media screen and (max-width: 1370px){ | ||
+ | .page-header{ | ||
+ | background-position-y:-15em; | ||
+ | } | ||
+ | } | ||
+ | </style> | ||
+ | <section class="page-header page-header-lg parallax parallax-3" style="background-image:url('https://static.igem.org/mediawiki/2016/d/dd/T--Valencia_UPV--protocolsTitleBack.png')"> | ||
+ | <div class="overlay dark-5"><!-- dark overlay [1 to 9 opacity] --></div> | ||
+ | <div class="container"> | ||
+ | <h1>Protocols</h1> | ||
+ | </div> | ||
</section> | </section> | ||
<section> | <section> | ||
− | <div class="container"> | + | <div class="container-fluid"> |
<div class="row"> | <div class="row"> | ||
<div class="col-md-2 col-sm-3"> | <div class="col-md-2 col-sm-3"> | ||
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"size-11 text-muted pull-right"></span>Petri | "size-11 text-muted pull-right"></span>Petri | ||
dish culture</a> | dish culture</a> | ||
+ | </li> | ||
+ | <li class="list-group-item"> | ||
+ | <a href= | ||
+ | "https://2016.igem.org/Team:Valencia_UPV/Notebook/Protocol#Liquidculture_id"> | ||
+ | <span class= | ||
+ | "size-11 text-muted pull-right"></span>Liquid culture</a> | ||
</li> | </li> | ||
<li class="list-group-item"> | <li class="list-group-item"> | ||
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<li class="list-group-item"> | <li class="list-group-item"> | ||
<a href= | <a href= | ||
− | "https://2016.igem.org/Team:Valencia_UPV/Notebook/Protocol# | + | "https://2016.igem.org/Team:Valencia_UPV/Notebook/Protocol#Protocol_id"> |
<span class= | <span class= | ||
"size-11 text-muted pull-right"></span>Digestion | "size-11 text-muted pull-right"></span>Digestion | ||
Line 295: | Line 312: | ||
<td>H20 milli-Q</td> | <td>H20 milli-Q</td> | ||
<td>225 mL</td> | <td>225 mL</td> | ||
+ | <td></td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
Line 325: | Line 343: | ||
<td>H20 milli-Q</td> | <td>H20 milli-Q</td> | ||
<td>484 mL</td> | <td>484 mL</td> | ||
+ | <td></td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
Line 348: | Line 367: | ||
<td>H20 milli-Q</td> | <td>H20 milli-Q</td> | ||
<td>49.4 mL</td> | <td>49.4 mL</td> | ||
+ | <td></td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
Line 427: | Line 447: | ||
<td>Adjust to pH=5.6 with KOH. | <td>Adjust to pH=5.6 with KOH. | ||
Sterilize</td> | Sterilize</td> | ||
+ | <td></td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
Line 599: | Line 620: | ||
<li><i>Agrobacterium</i>: rifampicin + the specific | <li><i>Agrobacterium</i>: rifampicin + the specific | ||
one for each construction.</li> | one for each construction.</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <p>To carry out this procedure is necessary to work in | ||
+ | the laminar flux cabinet.<br> | ||
+ | This procedure is necessary to made it in the laminar | ||
+ | flux cabinet. After <i>E. coli</i> transformation, | ||
+ | cells are put into an Eppendorf letting them grow at | ||
+ | 37°C. The spread plate method is done with 50-40 ul of | ||
+ | this bacteria culture. To plate it is used aIt is used | ||
+ | for plating a glass dipstick. After that, <i>E. | ||
+ | coli</i> culture plates are put at 37°C for | ||
+ | approximately 16h. <i>Agrobacterium</i> growth needs | ||
+ | 32h at 28°C.<br> | ||
+ | <br></p> | ||
+ | </div> | ||
+ | <div class="blog-post-item" id="Liquidculture_id"> | ||
+ | <h3>Liquid culture</h3> | ||
+ | <p>After competeted bacteria (E.coli DH5alpha) has been transformed and plated, they must be incubating at 37°C at least 16 hours. Then, a single colony must be picked up and insert it into liquid culture.<br>This starter culture is composed by:</p> | ||
+ | <ul> | ||
+ | <li>LB medium: Usually, 4 mililitres are used in E.coli cultures whereas in Agrobacterium cultures, 5 mililitres is required.</li> | ||
+ | <li>Antibiotics: Each plasmid of Golden Braid assemble offers different types of resistance to each bacteria culture. Antibiotics must be added at 1:1000 dilution. | ||
+ | <p></p> | ||
+ | <div class="table-responsive" style= | ||
+ | "width:55%;overflow:inherit"> | ||
+ | <table class= | ||
+ | "table table-bordered table-striped"> | ||
+ | <tr> | ||
+ | <th>Plasmid</th> | ||
+ | <th>Antibiotics</th> | ||
+ | </tr> | ||
+ | <td>pUPD2</td> | ||
+ | <td>Ampicilin</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>α</td> | ||
+ | <td>Kanamycin</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Ω</td> | ||
+ | <td>Spectinomycin</td> | ||
+ | </table> | ||
+ | </div> | ||
+ | <p></p> | ||
+ | </li> | ||
+ | |||
</ul> | </ul> | ||
<br> | <br> | ||
Line 709: | Line 775: | ||
</table> | </table> | ||
</div> | </div> | ||
− | <p></p> | + | <br><p>In order to carry out a ligate reaction, This protocol must be followed:</p> |
+ | <ol> | ||
+ | <li> Maximum number of cycles are 50</li> | ||
+ | <li> seg 37ºC during 2 minutes</li> | ||
+ | <li> seg 16,0ºC during 4 minutes</li> | ||
+ | <li> Fin extn 65.0ºC during 20 minutes</li> | ||
+ | <li> Fin hold 12ºC</li> | ||
+ | </ol> | ||
</div> | </div> | ||
<div class="blog-post-item" id="DigestionProtocol_id"> | <div class="blog-post-item" id="DigestionProtocol_id"> | ||
Line 766: | Line 839: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>α1</td> |
<td>EcoRI</td> | <td>EcoRI</td> | ||
<td>Specific</td> | <td>Specific</td> | ||
</tr> | </tr> | ||
+ | <tr> | ||
+ | <td>α2</td> | ||
+ | <td>HindIII</td> | ||
+ | <td>Specific</td> | ||
+ | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>Ω1</td> |
<td>BamHI</td> | <td>BamHI</td> | ||
<td>Specific</td> | <td>Specific</td> | ||
</tr> | </tr> | ||
+ | <tr> | ||
+ | <td>Ω2</td> | ||
+ | <td>EcoRV</td> | ||
+ | <td>Specific</td> | ||
+ | </tr> | ||
</table> | </table> | ||
</div> | </div> |
Latest revision as of 19:38, 19 October 2016
Protocols
Assembly workflow
This is the general assembly workflow that is followed by our team. It is based on the GoldenBraid assembly method
Orange DNA Genome Extraction Protocol (Adapted from Delaporta extraction protocol)
DNA EB Extraction Buffer | DNA EB Extraction Buffer |
---|---|
100mM Tris pH 8 | 50ml of Tris HCl 1M |
50mM EDTA pH 8 | 50 ml of EDTA 0.5M pH8 |
500 mM NaCl | 14.63 g NaCl |
Raise to 0.5 L | Raise to 0.5L |
When it is going to be used, add b-mercaptoethanol to 10mM (10μL).
First phase
- Weight 1g of tissue, freeze and grind with liquid nitrogen. Turn on water bath to 65°C
- Add 15mL of extraction buffer (remember to add mercaptoethanol, 10μL) in a beckman tube.
- Add 1mL of SDS 20%, shake vigorously and incubate 10 minutes at 65°C. Shake time to time.
- Add 5mL of K-Ac 5M, shake vigorously and incubate 0°C during 20 minutes.
- Centrifuge, JA20, 14krmp, 20 minutes.
- Filter the supernatant through a nylon fabric of 30u and pass to clean beckman tubes.
- Add 10mL of isopropanol, mix and incubate at -20°C during minimum 20 minutes. They can be left overnight.
- Centrifuge, JA20, 10krmp, 15 minutes.
- Pour and dry partially with the tubes inverted on blotting paper during 10 minutes. Warning: occasionally, the pellet may slide, so it is convenient to control the dry process.
- Resuspend the pellet in 0.7mL of TE (Tris HCl 50mM + EDTA 10mM). Put on Eppendorf tubes. Centrifuge 10 minutes at 7000rmp.
- Transfer the supernatant to other Eppendorf. Add 75μL of Na-Ac 3M (approximately 1/10 of the volume) and 0.5mL of isopropanol (around 60% of volume). Mix well and centrifuge 2-3 minutes at 10000rpm. If no pellet is produced, it will be a DNA concentration in the bottom; in this case, the supernatant should be removed with a pipette being extremely careful to not lose the DNA.
- Wash the sediment once or twice with 70% ethanol (200-100 μL and centrifuge). Dry on speed-vac and resuspend in 0.1mL of mili-Q water. If with this amount of water it is not well dissolved, add more water each time. To dissolve it is left on ice, shaking from time to time, during approximately one hour.
Second phase
- Add RNAase A up to 100 ug/L (add V/99 of the stock solution at 10mg/mL), digest at 37°C at least during 20 minutes.
- Add 1 volume of phenol and invert during 1 minute.
- Add 1 volume of chloroform. Centrifuge 5 minutes.
- Transfer upper aqueous phase to other Eppendorf. Add NaCl 5M until 0.2M (V x 0.0416 of NaCl 5M)
- Centrifuge 15 minutes. If centrifuged is not clear, add 100 ug of ethanol and then, centrifuge 15 minutes.
- Wash with ethanol 70%. Dry with speed-vac and dissolve in 0.1 ml of H2O milli-Q.
Rice DNA Genome Extraction Protocol
- Prepare buffers, cool centrifuge at 4°C and heat up thermoblock at 50°C and 37°C.
- Put around 0.2 gr of vegetal material in an Eppendorf.
- Add, previously mix a and b:
-
- 0.55 mL of Extraction Buffer (mash and put directly the buffer. Put it on ice. A pasty green will stay.
- 0.55 mL of phenol (tris-HCl) / chloroform / isoamyl (25/24/1)
- Vortex 20 seconds and put on ice.
- Centrifuge 5 minutes at maximum speed (4°C). Two phases will be formed.
- Transfer the supernatant (around 500 ug) to other Eppendorf.
- Add 0.55 mL of phenol (tris-HCl) / chloroform / isoamyl (25/24/1)
- Vortex 20 seconds and put on ice.
- Centrifuge 5 minutes at maximum speed (4°C). Two phases will be formed. Collect upper phase into a new Eppendorf.
- Add 0.1 volume of AcNa 3M and 2.5 volumes of EtOH absolute. Put it on ice and mash it manually.
- Centrifuge 10 minutes at maximum speed (4°C)
- Pour liquid and let dry between 30-60 minutes (cover opened)
- Add 200μL of TNE and 2 ug of RNAsa (10mg/mL stock)
- Incubate 10 minutes at 37°C
- Resuspend carefully pellet and spin to let fall liquid from walls.
- Add 200 ug of phenol (tris-HCl)/ chloroform/isoamyl (25/24/1). Slowly invert it.
- Centrifuge 5 minutes at maximum speed.
- Transfer the supernatant (around 200 ug) to other Eppendorf.
- Add 50-100ug of H2O milli-Q. Then, add 0.1 Volume of AcNa 3M + 2 Volume of EtOH 100%. Invert it.
- Let it:
-
- Overnight at -20°C
- 20 minutes at -80°C
- Centrifuge 30 minutes at maximum speed. Pour liquid and let pellet dries.
- Add 100ug of EtOH 70% and put it quickly on ice. Once they are all, shake it.
- Centrifuge 5 minutes at maximum speed.
- Pour liquid.
- Resuspend pellet in H2O milli-Q
Extraction Buffer | Stock solution | Volume for 500mL |
---|---|---|
0.2M Tris-HCl, ph=9.0 | 1M Tris-HCl, pH=9.0 | 100 mL |
0.4LiCl | 2M LiCl | 100 mL |
25mM EDTA | 0.5M EDTA | 25 mL |
1% SDS | 10% SDS | 50 mL |
H20 milli-Q | 225 mL |
TNE Buffer | Stock solution | Volume for 500mL |
---|---|---|
0.01M Tris-HCl, pH=8 | 1M Tris-HCl, pH=8 | 5 mL |
0.1M NaCl | 5M NaCl | 10 mL |
1mM EDTA, pH=8 | 0.5M EDTA | 1 mL |
H20 milli-Q | 484 mL |
TE Buffer | ||
---|---|---|
10mM Tris pH=8 | 1M Tris pH=8 | 0.5 mL |
1mM EDTA | 0.5M EDTA | 0.1 mL |
H20 milli-Q | 49.4 mL |
Phenol (Tris-HCl)/Chloroform/isoamyl 25/24/11 |
---|
AcNA 3M |
RNAsa |
EtOH Absolute. |
Agrobacterium infiltration
- Centrifuge 15 minutes at 3000 rpm the Agrobacterium culture.
- Prepare infiltration medium:
- 10 mL of MES 10X
- 1 mL of MgCl 100X
- Raise to 100 mL
- Weight 9.8 mg of Acetosyringone and resuspend it in 250 μL of DHSO. Add 100 μL to the 100mL previously made.
- Discard supernatant and resuspend in 5mL of Agroinfiltration medium.
- Vortex to mix correctly
- Cover falcons tubes with foil and incubate for 2 hours with stirring.
- Measuring OD
- C0 x V0 = C x V V = 20 mL C = 0.2 g/mol
- V0 = (0.2 x 20)/(0.38 x 5)=2.11 mL of culture is necessary to obtain the required optical density.
MES 10X | Volume | Notes |
---|---|---|
H2O milliQ | 500mL | Adjust to pH=5.6 with KOH. Sterilize |
MES | 10.66 g | Adjust to pH=5.6 with KOH. Sterilize |
Acetosyringone 20Mm(1000X) | Adjust to pH=5.6 with KOH. Sterilize |
Volume | Notes | |
---|---|---|
Acetosyringone | 78.48 mg | - |
DMSO | 2mL | - |
MgCl2 (100X) | Volume | Notes |
---|---|---|
MgCl2 | 20.3 g | Sterilize |
H2O | Until 100mL | Sterilize |
E.coli Transformation
Electroporation method was used in order to transform a DNA construction. This procedure is commonly used for E. coli and Agrobacterium.
- The electroporation cuvette was put in ice 10 minutes before inserting the cells. An aliquot of electrocompetent cells are taken out of the -80°C freezer, and they are put immediately into ice.
- 1-2 ul of the ligation product are taken and added carefully to them.
- 60 ul of the mix are taken and put into an electroporation cuvette making sure that there are no bubbles.
- The cuvette is dried and put in the electroporator, making sure that any spark is done. In that case, the process does not work and must be repeated. This could happen when plasmid and E. coli concentration are not optimal.
- The voltage for the electroporator is 1500V for E. coli and 1440V for Agrobacterium.
- Transformed cells are resuspended in 300 μL of LB in the electroporation cuvette. Then, they are taken and put into an Eppendorf letting them grow in the shaker for 2 hours at 37°C.
Minipreps
In order to carry out all of necessary Miniprep
-extraction of the plasmids out of E. coli -the
protocol of the Omega kit (Plasmid DNA Mini Kit I Spin
Protocol) is used. The steps to do it are:
- Grow 1-5 mL culture overnight in a 10-20 mL culture tube.
- Centrifuge at 10.000 x g for 1minute at room temperature. Decant or aspirate and discard the culture media.
- Add 250 ul Solution I mixed with RNase A. Vortex or pipet up and down to mix thoroughly. Transfer suspension into a new 1.5mL microcentrifuge tube.
- Add 250 ul Solutions II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary. Avoid vigorous mixing and do not exceed a 5 minute incubation.
- Add 350 ul Solution III. Immediately invert several times until a flocculent white precipitate forms. Centrifuge at maximum speed (>13.000xg) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
- Insert a HiBind DNA Mini Column into a 2 mL Collection tube.
- Transfer the cleared supernatant from Step 6 CAREFULLY aspirating it into the HiBind DNA Mini Column. Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse the collection tube.
- Add 500 ul HBC Buffer diluted with isopropanol
- Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse collection tube.
- Add 700 ul DNA Wash Buffer diluted with ethanol. Centrifuge at maximum speed for 1 minute. Discard the filtrate and reuse the collection tube.
- Centrifuge the empty HiBind DNA Mini Column at maximum speed for 2 minutes to dry the column. This step is critical for removal of trace ethanol that may interfere with downstream applications.
- Transfer the HiBind DNA Mini Column into a nuclease-free 1.5 mL microcentrifuge tube.
- Add 50 ul Elution Buffer or sterile deionized water directly to the center of the column membrane.Let sit at room temperature for 1 minute. Centrifuge at maximum speed for 1 minute.
- Store eluted DNA at -20°C.
Petri dish culture
Bacteria are transformed with a plasmid which gives
them resistance to a specific antibiotic. Petri dishes
must include this antibiotic to reject those bacteria
that have not acquired the plasmid.
- 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.
To carry out this procedure is necessary to work in
the laminar flux cabinet.
This procedure is necessary to made it in the laminar
flux cabinet. After E. coli transformation,
cells are put into an Eppendorf letting them grow at
37°C. The spread plate method is done with 50-40 ul of
this bacteria culture. To plate it is used aIt is used
for plating a glass dipstick. After that, E.
coli culture plates are put at 37°C for
approximately 16h. Agrobacterium growth needs
32h at 28°C.
Liquid culture
After competeted bacteria (E.coli DH5alpha) has been transformed and plated, they must be incubating at 37°C at least 16 hours. Then, a single colony must be picked up and insert it into liquid culture.
This starter culture is composed by:
- LB medium: Usually, 4 mililitres are used in E.coli cultures whereas in Agrobacterium cultures, 5 mililitres is required.
- Antibiotics: Each plasmid of Golden Braid assemble offers different types of resistance to each bacteria culture. Antibiotics must be added at 1:1000 dilution.
Plasmid Antibiotics pUPD2 Ampicilin α Kanamycin Ω Spectinomycin
To carry out this procedure is necessary to work in
the laminar flux cabinet.
This procedure is necessary to made it in the laminar
flux cabinet. After E. coli transformation,
cells are put into an Eppendorf letting them grow at
37°C. The spread plate method is done with 50-40 ul of
this bacteria culture. To plate it is used aIt is used
for plating a glass dipstick. After that, E.
coli culture plates are put at 37°C for
approximately 16h. Agrobacterium growth needs
32h at 28°C.
Ligation reaction
The assembly of DNA fragments, commonly referred to
as Golden Gate assembly, is an efficient technique
where multiple inserts could be assembled into a vector
backbone. The net result is the ordered and seamless
assembly of DNA fragments in only one reaction. The
ligations have a total volume of 10 μL so all of
sequences that form part of the constructions are mixed
up in an Eppendorf of 0.2mL.
This mix is put in a thermocycler with the programs GB
(Golden Braid) or GG (Golden Gate).
Reagent | Volume (μL) |
---|---|
DNA fragments | 1 of each part of the assembly |
Plasmid (pUPD2 - α1 - α2) | 1 |
BSA 10X | 1.2 |
Ligase Buffer | 1.2 |
Bsmb I | 1 |
T4 ligase | 1 |
H2O milli-Q | Raise until final volume (10 μL) |
DNA1 in pUPD2 + DNA2 in pUPD2 = DNA1 + DNA2 in α | DNA1 in α1 + DNA2 in α2 = DNA1 + DNA2 in Ω |
---|---|
1 μL DNA1 in pUPD2 | 1 μL DNA1 in α1 |
1 μL DNA2 in pUPD2 | 1 μL DNA2 in α2 |
1 μL of α plasmid | 1 μL of Ω plasmid |
1.2 μL buffer ligase | 1.2 μL buffer ligase |
1.2 μL BSA10X | 1.2 μL BSA10X |
1 μL T4 ligase | 1 μL T4 ligase |
1 μL Bsmb I | 1 μL Bsa I |
4.6 μL H2O milli-Q | 4.6 μL H2O milli-Q |
In order to carry out a ligate reaction, This protocol must be followed:
- Maximum number of cycles are 50
- seg 37ºC during 2 minutes
- seg 16,0ºC during 4 minutes
- Fin extn 65.0ºC during 20 minutes
- Fin hold 12ºC
Digestion Protocol
One or more restriction enzymes are used to digest
the DNA, resulting in either non-directional or
directional insertion into the compatible plasmid. DNA
was obtained after doing athe Miniprep (Plasmid DNA
Mini Kit I Spin Protocol). For carrying out the
digestion to check if desired fragment is inside the
plasmid it is necessary to mix up the followingnext
components in a 200 ul Eppendorf.
Reagent | Volume (μL) |
---|---|
DNA | 3 |
Specific buffer | 1 |
Specific enzyme | 1 |
H2O milli-Q | 5 |
These are the specific enzymes and buffers for each
type of plasmid. The desired insert size for the clone
library determines which enzymes are selected, as well
as the digestion conditions. Generally, once the mix is
done, it is necessary to incubate it at 37°C at least 1
hour.
Plasmid | Enzyme | Buffer |
---|---|---|
pUPD2 | Not I | Orange |
α1 | EcoRI | Specific |
α2 | HindIII | Specific |
Ω1 | BamHI | Specific |
Ω2 | EcoRV | Specific |
Agarose gel electrophoresis
- Prepare Agarose gel Agarose gel is used in a concentration of 1% to separate medium size fragments. The gel is made with agarose powder, TAE buffer 1X and ethidium bromide. It is necessary to adjust the amount of agarose to get the desired gel concentration. For example, with 1g of agarose / 100 mL of TAE 1X, the gel concentration will be 1%. The ethidium bromide concentration is 1:1000 in TAE Buffer (In the previous example, 1 μL is needed). Small gels where 10 samples can run need:
- Weight 0.45 g of agarose in a scale precision.
- Measure 45mL of TAE 1X. Add both of them into an Erlenmeyer flask.
- Put it in a microwave for 1-3 min (until agarose is dissolved)
- Let agarose cool down.
- Add 0.45 μL of Ethidium bromide. This step is optional and allows the visualization of the DNA under UV light.
- Collocating agarose solution in the correct tray. Do not forget putting the electrophoresis comb. Pour slowly to avoid bubbles due to they could damage the gel.
- Let it at room temperature for 20-30 minutes.
- Running Agarose Gel:
-
- When agarose gel is solidified, put it into electrophoresis unit. It has to be covered by 1X TAE buffer.
- Load the molecular weight ladder. It allows to check if our fragment belongs to a specific band checking the size that it has.
- Load samples into the remaining wells. It is recommendable to put 5 μL of DNA sample with 1 μL of loading buffer.
- Run the gel at 100V (Small gel) or 120V (Big gel). The time varies depending on gel concentration and voltage.
- Visualize DNA fragments using a transilluminator.
Reagent | Amount |
---|---|
Agarose powder | 0.45 g |
TAE 1X | 45 mL |
Ethidium bromide | 0.45 μL |
Electrophoresis parameters | Electrophoresis parameters |
---|---|
DNA fragment | 5 μL |
Loading buffer | 1 μL |
Molecular marker 1Kb | 5 μL |
Voltage | 100V to 120V |
Luciferase assay
This procedure is done with the PrΩ 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 -80°C.
The steps to follow are:
- The Passive lysis buffer 1x is prepared. Each disk of leaf needs 200ul. 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.
- Cut off two little leaf disks of approximately 0.8cm 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.
- Add 150ul of passive lysis buffer 1x to each Eppendorf tube.
- Vortex gently.
- Centrifuge 13200rpm during 15 minutes at 4°C. While switch on the luminometer.
- Dilute 2:3 the extracts on a new Eppendorf tube. Add 36ul of Passive lysis buffer 1x and 24ul of sample.
- Take an optimal plate to use in the luminometer. Fill luminometer wells with 40 ul of LUCII which is stored at -20°C.
- 10 ul of sample is added in each well. Wait 10minutes. During this time turn on and configure the luminometer.
- Measure luciferase activity
- Prepare 40 ul/well of Dual Glo 1x (STOP solution + substrate). The substrate is at 50x concentration and stored at -20°C.
- When the first luciferase measure is done, it is necessary to add 40 ul of Dual Glo into each well. Let it rest during 10 minutes.
- Measure the Renilla activity.
- Take the obtained information and analyze it.
It must be kept in mind that 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.