Preparation of Primers
When the primers arrive, we give them a spin to settle any residue on the lid. We then dissolve the powder very well in water that has been recently sterilized and opened at that time. In addition, we also use new tips. We follow the instructions given in the leaflet that comes with the primers so that the final concentration is 100 μM. From the stock solutions we make aliquots with a concentration of 10 μM. We keep them at -20°C.
Preparation of dried DNA
If the DNA is dry we do a spin. Then we redissolve it well under a flame. To assist the redissolution we place it in a water bath at 30°C for 5 min. Afterwards, we keep it on ice. To store it, put it in the refrigerator.
Starting a liquid culture of bacteria
The various bacterial strains as well as bacteria carrying specific plasmids are stored in glycerol stocks at -80°C.
- You will need a gas, lighter, LB (lysogenic broth), falcon tubes, ampicillin (in a waterbath to defrost faster), and tips which you only use under a flame (note this on the box).
- First, we prepare in a falcon tube the entire amount of LB we will need by adding ampicillin so that its final concentration in the solution is 100 μg/ml.
- Add the appropriate amount of LB to the falcon tubes. Always make sure that there is enough ventilation in the tube. For this purpose, the volume remaining free in the tube should be at least a little more than twice the volume of LB added (e.g. in a 15-well facon we put up to 4 ml LB).
- We take ice and put in the appropriate glycerol stocks. In case we want more than two different samples, we take them out of the freezer half and half so they don't stay out for a long time.
- We work under a flame.
- The tips should be located to the right of the gas and opened so that they face the flame.
- Open the falcon tube and place the lid on the gas under the flame.
- Open the eppendorf carefully to avoid droplets.
- Put tip in a pipette (200 or 20), put it in the eppendorf tube, shake it around to get a small amount, and pour it all into the LB in the falcon tube.
- Close the lid after passing it over the flame.
- Place the falcon tubes in a beaker and add additional crinkled paper, making sure they do not shake. Cover the beaker with foil and tape around it with cellophane.
- E. coli are incubated overnight at 200 rpm and 37°C, while Agrobacterium tumefaciens are incubated at 200 rpm at 28°C.
Golden Gate Cloning
For the assembly you will need quantified aliquots of DNA containing each assembly piece, as well as the vector.
If assembly pieces are cloned in level 0 vectors (plasmids) use 100ng of the vector backbone and EQUIMOLAR* amounts of the other assembly pieces to a 15μl total volume assembly reaction mixture
Backbone vector: pBluescript\II\SK(-)\GG [BBa_K3935000] Use 10μl for transformation of E. coli competent cells and plate for Ampicillin selection. Also, selection can be made using xGal as this vector contains a lacZ gene.
Transformation of competent cells
A total of 10 μL of the ligation reaction was transformed into chemically competent DH10B cells. After incubation at 4°C for 30 min, the cells were heat-shocked at 42°C for 1 min and returned immediately to 4°C for 1 min. Then, LB was added up to a total volume of 1 mL. After incubation at 37°C with shaking at 200 rpm for 1 h, the cells were centrifuged at 2100 × g for 2 min, the supernatant was discarded, and the pellet was resuspended in the remaining solution, plated on solid LB with ampicillin (100 μg/mL) and incubated at 37°C overnight.
Isolation of plasmid DNA
Alkaline Lysis Buffer I:
Glucose 50 mM
Tris-Cl (pH 8.0) 25 mM
EDTA (pH 8.0) 10 mM
It is prepared whenever the previous one is finished and kept in the refrigerator.
Alkaline Lysis Buffer II:
NaOH 0.2 N
SDS 1% w/v
Make it every time, unless it is reused within a month, and keep it at room temperature.
Alkaline Lysis Buffer III:
Potassium acetate 5 M: 60 ml
Glacial acetic acid: 11.5 ml
H2O: 28.5 ml
It is kept in the refrigerator.
We will need a rack of tips to pour the liquid waste, a rack for the tubes, eppendorf tubes, the solutions listed above, ice, 70% EtOH, and 100% EtOH.
Cultures of E. coli in LB were incubated overnight at 37°C, 200 rpm until saturation (concentration more than 109 cells/mL). The saturated cultures were centrifuged at 11000 × g, 25°C, for 30 s. The supernatant was discarded, and the pellet was resuspended by vortexing in 100 μL alkaline lysis solution I, pH 8.0 (glucose 50 mM, Tris-HCl 25 mM, EDTA 10 mM, 4°C) per 3 mL of culture. Then, 200 μL of alkaline lysis solution II (NaOH 0.2 N, SDS 1% w/v, 25°C) and 150 μL of alkaline lysis solution III (60 mL potassium acetate 5 M, 11.5 mL glacial acetic acid, 28.5 mL H2O, 4°C) were added per 3 mL of culture. Each sample was mildly mixed after the addition of solutions II and III. After incubation at 4°C for 3 min, it was centrifuged at 13100 × g for 5 min at 4°C. The supernatant was transferred to a clean tube, and the DNA was precipitated with two volumes of absolute ethanol by shaking, incubating at 25°C for 2 min, and centrifuging at 13100 × g for 15 min. The supernatant was discarded, and the pellet washed with the addition of 500 μL 70% v/v ethanol and centrifuged at 13100 × g for 5 min. Finally, the ethanol was removed, and the pellet was dried and resuspended in 30 μL sterile distilled water (dH2O).
PCR with Phusion® HighFidelity- DNA Polymerase
Template DNA 1 ng: 1 μl
5X Phusion Buffer: 10 μl
Fw Primer (0.5 μM): 2.5 μl
Rv Primer (0.5 μM): 2.5 μl
dNTPs (0.2 mM): 1 μl
Phusion DNA Polymerase (2 U/μl): 0.5 μl
H2O: 32.5 μl
Total: 50 μl
Gel extraction and purification of PCR products
- Turn on the water bath at 48°C.
- We use a different scalpel for each sample and transfer each piece of gel to a different eppendorf tube. The tube is grasped without a glove to avoid transferring ethidium.
- For every 100 mg of ≤ 2% w/v agarose gel, 200 μl of NTI Buffer is added and the samples are incubated at 48°C until the gel is completely melted. At intervals, assist the dissolution with vortex. We then follow the NucleoSpin® Gel and PCR Clean-up protocol.
- The final elution is performed with 30 μl H2O.
Template DNA (all the amount isolated): 30 µl
10X Taq DNA Polymerase Buffer: 5 μl
dATPs (0.5 mM): 2.5 μl
Taq DNA Polymerase (5 U/μl): 0.2 μl
ddH2O: 12.3 μl
Total: 50 μl
- Incubate at 72°C for 30 min (PCR machine).
- Quantify by loading 1 μl of DNA, 1 μl of Orange G, and 4 μl of H2O onto the gel mixture and running it at 100 V for 15 min.
The amount of DNA insert needed is calculated from the following formula:
The amount of vector used is 50 ng and the sizes of the vector and insert DNA in bp are also known. Usually the vector/insert ratio is 3 molecules of insert for one molecule of vector (3:1). Thus, the only unknown quantity is the amount of insert DNA.
10X T4 DNA Ligase Buffer: 2 μl
vector DNA (50 ng): 1 μl (pICSL86922)
insert DNA (minsert): 59.4 ng
H2O: __ (so that the final volume is 20 µl)
T4 DNA Ligase: 1 μl
Total: 20 μl
- Incubate at room temperature for 2 h.
Transformation of Agrobacterium Tumefaciens
Preparation of Competent Cells
1. Inoculate 200 ml of LB media with 1 ml of an overnight culture of the chosen strain of Agrobacterium. Incubate at 28ºC with vigorous agitation.The culture was created in the late afternoon and harvested the next morning
2. The cells were grown to log phase (OD 550 0.5-0.8).
3. Pellet the cells in a benchtop centrifuge at 5000 x g for 10 minutes at room temperature.
4. Wash the pellet with sterile 1X TE.
5. Resuspend the cells in 0.1X the original volume of LB, and aliquot 250μl fractions in microcentrifuge tubes.
6. Snap-freeze in liquid nitrogen and store at -70ºC.
7. Thaw competent Agrobacterium on ice (use 250 μl per transformation reaction), and add DNA (up to 10 μl, 100-1000ng) and flick tube gently to mix.
8. Keep the mixture on ice for 5 minutes, and then transfer to liquid nitrogen for 5 minutes.
9. Incubate the mixture for an additional 5 minutes in a 37ºC water bath.
10. Transfer 250 μl of cells to a 15 ml Corning tube containing 1 ml of LB and shake at 28ºC for 2 hours.
11. Collect the cells by spinning 2 min at 5000 rpm, and resuspend cells in 100-200 µl LB. Spread them on two LB agar plates containing the ampicillin(100 μg/mL).
12. Incubate the cells for 2 days at 26-28ºC for colonies to form.
The solution is placed in a needleless syringe for injection(,without a needle). The syringe tip is placed against the underside of a leaf while mild counter pressure is applied to the opposite side of the leaf. The Agrobacterium suspension is then injected into the leaf's airspaces via stomata, or sometimes a small incision on the underside of the leaf.