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Fig. 9 | Plant Methods

Fig. 9

From: A cell death assay in barley and wheat protoplasts for identification and validation of matching pathogen AVR effector and plant NLR immune receptors

Fig. 9

Overview of steps for the transfection of isolated protoplasts. Step 18: 300 µl Transfection Buffer 1 containing protoplasts are transferred to each transfection tube. Step 19: Using a pipette, plasmid transfection sample 1 is added to transfection sample tube 1 directly into Transfection Buffer 1 containing protoplasts. Step 20: 350 µl Transfection Buffer 2 is added to transfection sample tube. Step 21: Transfection sample tube is inverted 12 times. Step 22: Transfection sample tube is placed into rack in the dark. Step 23: Steps 19 to 22 are repeated with all other transfection samples one after another. Step 24: After 15 min incubation in the dark, 2× 660 µl of Wash Buffer is added to transfection sample tube. Step 25: Transfection sample tube is inverted 8 times. Step 26: All transfection sample tubes (up to six at the time) are centrifuged at 100×g for 3 min. Step 27: Using a pipette, 2× 965 µl are removed from all transfection sample tubes. Step 28: 965 µl of Regeneration Buffer is transferred into each transfection sample tube. Step 29: All transfection samples tubes containing protoplasts are regenerated at 20 °C in the dark for 16 h

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