| 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:
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
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.
Luciferase assay protocol
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.
Western blot protocol
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.
Protoplasts protocol
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.
Protoplast luciferase assay protocol
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.
