Hi Panchsheela,

You haven't said what kind of competent cells you want to prepare. Are you going to use electroporation, triparental mating, heat shock...? The methods vary depending on the application. For instance, if you want to make electrocompetents you must ensure that the cells are well washed, because remaining salts in the competents can cause arcing in the electroporator and effectively fry the cells.

I strongly recommend you to do two things:

- start everything from single isolated colonies, so you know that your culture is pure.

- once you have the competents, check them growing them on plates with a range of antibiotics for the selectable markers you have in the lab, e.g. Control: gent and rif (this should grow normally), test kanamycin (nothing should grow), test spectinomycin (nothing should grow), etc. If you see growth where it shouldn't, well... your cells contain an unwanted plasmids and you should discard them. Alternatively, you can do this before making the competents, by re-streaking the single colony in selective plates. If you use cells that contain unwanted plasmids you face a lot of trouble.

This is the protocol I normally use for electrocompetents, which does not differ much from the one above. You can use this protocol for any agro strain.

Materials:

• Single colony of Agrobaterium cells

• LB or YEP medium

• LB or YEP plates

• Antibiotics

• Chilled 50-ml or 250-ml polypropylene tubes

• Sterile 10% glycerol or 1mM HEPES (autoclaved, stored at 4 °C)

• 1.5 ml microcentrifuge tubes.

• Liquid nitrogen or dry ice.

Background and Notes

Agrobacterium strains have a chromosomal Rifampicin resistance.

- GV3101 pMP90: Rif (chromosomal), Gent (Ti plasmid). Appropriate to transform Arabidopsis, tomato, tobacco.

Abbreviatures: Rif: rifampicin 20 mg/L, Gent: gentamycin 25 mg/L

All materials and reagents coming into contact with bacteria should be sterile. It is critical that once the cells are cold, they stay cold. Also, be thorough with the washes and resuspend the pellet fully -otherwise salts in the electroporation cuvette will cause arching and low or nil electroporation efficiencies.

Protocol to make competent cells

1. Streak desired strain on an LB or YEP plate with the appropriate antibiotics. Incubate at 28°C for two days.

2. Grow a single colony O/N in 100 ml LB or YEP with antibiotics in a shaking incubator at 28°C until it reaches an OD600 of 0.5.

3. Transfer to two sterile 50 ml conical tubes

4. Spin down for 20 min at 4000 rpm at 4°C. Discard supernatant.

5. Resuspend in cold (4°C) 10% glycerol and repeat step 4 further two times.

6. Resuspend each pellet in 1 ml cold 10% glycerol.

7. Make 50-80 µl aliquots, snap-freeze in liquid nitrogen and store at -80 for up to 6 months.

Protocol for Electroporation:

1. Keep competent cells on ice and chill an electroporation cuvette.

2. Add 1 µl of plasmid (20 - 500 ng) to the agrobacterium aliquot, mix gently and transfer to the pre-chilled cuvette.

3. Dry the exterior of the cuvette with a bit of paper and insert in the electroporator.

4. Put the lid on, adjust voltage to 1800 V, press start and wait till you hear the 'beep'*. Immediately take the cuvette and add 500 µl of SOC or LB with no antibiotics.

5. Transfer to a microcentrifuge tube and rescue for 3-4 h in a shaking incubator at 28 °C.

6. Plate out 25-100 µl in a plate with the appropriate antibiotics and incubate at 28 °C for two days.

* If you hear a 'bang!' your cuvette has arced and your cells are mostly dead. Make sure that the cuvette is dry, or use a different agrobacterium stock that has been properly washed)

The Agrobacterium tumefaciens strain LB4404 was grown in 10ml YEP broth at 300rpm

at 30qC for 24 hours. A flask containing 1.0 liter YEP broth was inoculated with the grown culture

and placed on an orbital shaker at 350rpm at 30qC for 16 hours. The OD value was recorded at

600 to confirm the optimal bacterial growth (0.6 to 0.8). The culture was cooled down at 4°C and

transferred to 50 ml culture tubes. The cells were harvested through 15 minutes centrifugation at

4000rpm at 4qC. The pellets were twice washed with 1M HEPES solution. Again wash it with 10

% glycerol. Finally each pellet was dissolved in 1.0ml 10% glycerol solution under ice-cold

conditions. The aliquots of 50Pl were prepared and stored at -70qC.

Thank you very much. You have mentioned the protocol for LBA4404 strain but i need to prepare for Agro GVGV3101 strain, so will this protocol remain same or there would be any kind of change ??

Hi Panchsheela,

You haven't said what kind of competent cells you want to prepare. Are you going to use electroporation, triparental mating, heat shock...? The methods vary depending on the application. For instance, if you want to make electrocompetents you must ensure that the cells are well washed, because remaining salts in the competents can cause arcing in the electroporator and effectively fry the cells.

I strongly recommend you to do two things:

- start everything from single isolated colonies, so you know that your culture is pure.

- once you have the competents, check them growing them on plates with a range of antibiotics for the selectable markers you have in the lab, e.g. Control: gent and rif (this should grow normally), test kanamycin (nothing should grow), test spectinomycin (nothing should grow), etc. If you see growth where it shouldn't, well... your cells contain an unwanted plasmids and you should discard them. Alternatively, you can do this before making the competents, by re-streaking the single colony in selective plates. If you use cells that contain unwanted plasmids you face a lot of trouble.

This is the protocol I normally use for electrocompetents, which does not differ much from the one above. You can use this protocol for any agro strain.

Materials:

• Single colony of Agrobaterium cells

• LB or YEP medium

• LB or YEP plates

• Antibiotics

• Chilled 50-ml or 250-ml polypropylene tubes

• Sterile 10% glycerol or 1mM HEPES (autoclaved, stored at 4 °C)

• 1.5 ml microcentrifuge tubes.

• Liquid nitrogen or dry ice.

Background and Notes

Agrobacterium strains have a chromosomal Rifampicin resistance.

- GV3101 pMP90: Rif (chromosomal), Gent (Ti plasmid). Appropriate to transform Arabidopsis, tomato, tobacco.

Abbreviatures: Rif: rifampicin 20 mg/L, Gent: gentamycin 25 mg/L

All materials and reagents coming into contact with bacteria should be sterile. It is critical that once the cells are cold, they stay cold. Also, be thorough with the washes and resuspend the pellet fully -otherwise salts in the electroporation cuvette will cause arching and low or nil electroporation efficiencies.

Protocol to make competent cells

1. Streak desired strain on an LB or YEP plate with the appropriate antibiotics. Incubate at 28°C for two days.

2. Grow a single colony O/N in 100 ml LB or YEP with antibiotics in a shaking incubator at 28°C until it reaches an OD600 of 0.5.

3. Transfer to two sterile 50 ml conical tubes

4. Spin down for 20 min at 4000 rpm at 4°C. Discard supernatant.

5. Resuspend in cold (4°C) 10% glycerol and repeat step 4 further two times.

6. Resuspend each pellet in 1 ml cold 10% glycerol.

7. Make 50-80 µl aliquots, snap-freeze in liquid nitrogen and store at -80 for up to 6 months.

Protocol for Electroporation:

1. Keep competent cells on ice and chill an electroporation cuvette.

2. Add 1 µl of plasmid (20 - 500 ng) to the agrobacterium aliquot, mix gently and transfer to the pre-chilled cuvette.

3. Dry the exterior of the cuvette with a bit of paper and insert in the electroporator.

4. Put the lid on, adjust voltage to 1800 V, press start and wait till you hear the 'beep'*. Immediately take the cuvette and add 500 µl of SOC or LB with no antibiotics.

5. Transfer to a microcentrifuge tube and rescue for 3-4 h in a shaking incubator at 28 °C.

6. Plate out 25-100 µl in a plate with the appropriate antibiotics and incubate at 28 °C for two days.

* If you hear a 'bang!' your cuvette has arced and your cells are mostly dead. Make sure that the cuvette is dry, or use a different agrobacterium stock that has been properly washed)

Protocol ll remain the same for both the strains. This protocol is for transformation through Electroporation. As the Agrobacterium have think membrane so Transformation through this method is easy and more successful. If you don't have Elctroporator than you may use chemically competent cells and can proceed for heat shock method of transformation