Team:Valencia UPV/Notebook/Protocol

Index

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:
1. 0.55 mL of Extraction Buffer (mash and put directly the buffer. Put it on ice. A pasty green will stay.
2. 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:
1. Overnight at -20°C
2. 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:
1. 10 mL of MES 10X
2. 1 mL of MgCl 100X
3. Raise to 100 mL
4. Weight 9.8 mg of Acetosyringone and resuspend it in 250 μL of DHSO. Add 100 μL to the 100mL previously made.
5. Discard supernatant and resuspend in 5mL of Agroinfiltration medium.
6. Vortex to mix correctly
7. Cover falcons tubes with foil and incubate for 2 hours with stirring.

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

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.

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

Reagent	Amount
Agarose powder	0.45 g
TAE 1X	45 mL
Ethidium bromide	0.45 μL

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:
1. When agarose gel is solidified, put it into electrophoresis unit. It has to be covered by 1X TAE buffer.
2. Load the molecular weight ladder. It allows to check if our fragment belongs to a specific band checking the size that it has.
3. Load samples into the remaining wells. It is recommendable to put 5 μL of DNA sample with 1 μL of loading buffer.
4. Run the gel at 100V (Small gel) or 120V (Big gel). The time varies depending on gel concentration and voltage.
5. Visualize DNA fragments using a transilluminator.

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.