Culture preparation Grow the recipient strain overnight in YPD media at 30 °C with shaking at 160 rpm.
{"action": "grow", "output": "", "reagent": ["YPD media"], "length": [""], "device": [""]}
Harvest the culture in 50 ml tubes by centrifugation at 1,000 x g for 5 min. #####%%%%% output "sterile water" are used in line6
{"action": "harvest", "output": "sterile water", "container": ["50 ml tubes"], "device": ["centrifugation"], "speed": ["1,000 x g"], "time": ["5 min"]}
Wash the cell pellet(s) once with 25 ml of sterile water at room temperature.
{"action": "wash", "output": "", "reagent": ["sterile water"], "temperature": ["room temperature"], "time": [""]}
Treatment of cells Each cell pellet derived from 50 ml culture is very gently resuspended by slow pipetting in 8 ml sterile water,
{"action": "resuspend", "output": "", "volume": ["50 ml culture"]}
Incubate the cell suspension(s) for 30 min at 30 °C . #####%%%%% output "DTT" are used in line12
{"action": "incubate", "output": "DTT", "time": ["30 min"], "temperature": ["30 \u00b0C"]}
Add 250 µl of DTT .
{"action": "add", "output": "", "reagent": ["DTT"]}
incubate the suspension(s) for 60 min at 30 °C
{"action": "incubate", "output": "", "container": ["the suspension(s)"], "temperature": ["30 \u00b0"], "reagent": [""], "time": ["60 min"]}
Add 40 ml of sterile pre-cooled water at 1,000 x g for 5 min C.
{"action": "add", "output": "", "volume": ["40 ml"], "reagent": ["sterile pre-cooled water"]}
centrifuge the suspensions
{"action": "centrifuge", "output": "", "container": ["the suspensions"], "force": [""], "time": [""]}
Carefully resuspend cell pellets by slow pipetting in 25 ml of sterile water (4 °C) .
{"action": "resuspend", "output": "", "container": ["cell pellets"], "reagent": ["sterile water"], "volume": ["25 ml"], "temperature": ["4 °C"]}
repeat the centrifugation step
{"action": "repeat", "output": "", "reagent": [""]}
Carefully resuspend cell pellets in 5 ml pre-cooled 1 M sorbitol .
{"action": "resuspend", "output": "", "container": ["cell pellets"], "volume": ["5 ml"], "reagent": [""]}
repeat the centrifuging step
{"action": "repeat", "output": "", "reagent": [""]}
Finally, resuspend the pellets of electrocompetent cells in 550 µl of pre-cooled 1 M sorbitol.
{"action": "resuspend", "output": "", "concentration": ["pre-cooled 1 M sorbitol"], "volume": ["550 µl"]}
Add 5 to 10 µl of highly purified transforming DNA (1 to 3 µg), mix gently .
{"action": "add", "output": "", "volume": ["5 to 10 µl"], "reagent": ["highly purified transforming DNA (1 to 3 µg)"]}
incubate tubes on ice for 10 min #####%%%%% output "YPD media" are used in line34
{"action": "incubate", "output": "YPD media", "temperature": ["ice"], "time": ["10 min"]}
Add 950 µl of YPD media to each cuvette .
{"action": "add", "output": "", "reagent": ["YPD media"], "container": ["each cuvette"]}
transfer the suspension to a tube
{"action": "transfer", "output": "", "container": ["the suspension"]}
Add 45 µl of electrocompetent cell suspensions .
{"action": "add", "output": "", "volume": ["45 µl"], "reagent": ["electrocompetent cell suspensions"]}
incubate the plate for 10 min on ice
{"action": "incubate", "output": "", "container": ["the plate"], "temperature": ["ice"], "time": ["10 min"]}
Make sure that the DNA samples are at the bottom of the wells.
{"action": "make", "output": "", "length": [""]}
Cover the plate with a sterile lid . #####%%%%% output "YPD media" are used in line46
{"action": "cover", "output": "YPD media", "container": ["the plate"], "device": ["a sterile lid"]}
Transfer the cell suspension into a 96-well plate with 950 µl of YPD media.
{"action": "transfer", "output": "", "container": ["the cell suspension"], "reagent": ["YPD media"]}
The media in the plate can be used to flush the wells of the electroporation plate.
{"action": "use", "output": "", "volume": [""]}
Afterwards, centrifuge the tubes at 1,000 x g for 5 min. #####%%%%% output "sterile water" are used in line52
{"action": "centrifuge", "output": "sterile water", "container": ["the tubes"], "force": ["1,000 x g"], "time": ["5 min"]}
Discard supernatants in 100 µl of sterile water.
{"action": "remove", "output": "", "reagent": ["sterile water"]}