TITLE: Making electrocompetent Agrobacterium tumefaciens
AUTHORS: Johannes Wolfram Debler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol yields about 5 ml of electrocompetent </span><span style = "font-style:italic;">Agrobacterium tumefaciens </span><span>cells, aliquotted into 80 ul you get 50-60 tubes out of it.</span></div><div class = "text-block">Rifampicin is dark red and the colour will change over time, it does however NOT interfere with OD600 measurements, so you can use LB to blank. </div></div>

[STEPS]
?. [Things you need to prepare]
LB medium (for 1 litre combine 10 g Tryptone, 5 g Yeast Extrad and 10 g NaCl, adjust pH to 7.0 and autoclave)10% glycerol solution (ice cold)H2O (ice cold)liquid nitrogen50 - 60 x 1.5 ml tubes (pre-chill in fridge)10 ml pipette tips8 x 50 ml tubes (pre-chill in fridge)Your favourite strain of Agrobacterium tumefaciens grown on a plateDepending on your strain of Agrobacterium you'll need the following antibiotics:AGL1: rifampicin 100 ng/ulLBA 4404: rifampicin 100 ng/ul, streptomycin 100 ng/ul --> grows slow and clumpsGV3101::pMP90: rifampicin 100 ng/ul, gentamicin 25 ng/ul --> grows fastest of the threeDepending on your strain of Agrobacterium you'll need the following antibiotics:AGL1: rifampicin 100 ng/ulLBA 4404: rifampicin 100 ng/ul, streptomycin 100 ng/ul --> grows slow and clumpsGV3101::pMP90: rifampicin 100 ng/ul, gentamicin 25 ng/ul --> grows fastest of the three
?. [Day 1]
Inoculate 10 ml LB (containing the appropriate antibiotics) with 1 colony of Agrobacterium tumefaciens in the morning and incubate at 28°C at 250 rpm for the rest of the day.
28 °C
[LB]
?. [Day 1]
In the evening inoculate 3 x 150 ml LB (containing the appropriate antibiotics) with 3 ml each of the culture grown throughout the day.
28 °C
[LB]
?. [Day 1]
Incubate at 28°C at 250 rpm until OD600 reaches 0.8 - 1.0 (This usually takes overnight, depending on Agro strain im might take longer).
28 °C
?. [Day 2]
Check OD600, and combine all 3 cultures once OD600 reaches 0.8-1.0
?. [Day 2]
Chill culture by  putting it on ice for 15 minutes and swirling it regularly. From here on everything needs to happen on ice or at 4°C!
4 °C
?. [Day 2]
Distribute culture into 8 x 50 ml tubes and pellet by centrifugation at 3000 - 4000 x g for 20-30 minutes at 4°C.
4 °C
?. [Day 2]
Discard supernatant and resuspend each pellet in 10 ml ice cold H2O.
[H2O]
?. [Day 2]
Adjust volume to 50 ml each with ice cold H2O.
?. [Day 2]
Pellet cells by centrifugation at 3000 - 4000 x g for 20-30 minutes at 4°C.
4 °C
?. [Day 2]
Discard supernatant and resuspend each pellet in 10 ml ice cold H2O.
[H2O]
?. [Day 2]
Combine solutions into 4 x 50 ml tubes and adjust to 50 ml with ice cold H2O.
?. [Day 2]
Pellet cells by centrifugation at 3000 - 4000 x g for 20-30 minutes at 4°C.
4 °C
?. [Day 2]
Discard supernatant and resuspend each pellet in 10 ml ice cold H2O.
[H2O]
?. [Day 2]
Combine solutions into 2 x 50 ml tubes and adjust to 50 ml with ice cold H2O.
?. [Day 2]
Pellet cells by centrifugation at 3000 - 4000 x g for 20-30 minutes at 4°C.
4 °C
?. [Day 2]
Discard supernatant and resuspend each pellet in 10 ml ice cold H2O.
[H2O]
?. [Day 2]
Combine solutions into 1 x 50 ml tubes and adjust to 50 ml with ice cold H2O.
?. [Day 2]
Pellet cells by centrifugation at 3000 - 4000 x g for 20-30 minutes at 4°C. (Use a counterbalence tube)
4 °C
?. [Day 2]
Discard supernatant and resuspend pellet in 4.5 ml ice-cold 10% glycerol.
[10% glycerol]
?. [Day 2]
Dispense 80 ul aliquots into pre-chilled 1.5 ml tubes and flash freeze in liquid nitrogen.
?. [Day 2]
Store aliquots at -80°C until use.