BEFORE WE START -------------------------------------------------------------------------------- This protocol requires 3 96-well plates and 8 falcon tubes: Each plate will hold 200 uL per well Each tube will hold 9 mL We need each of the 8 species grown in monoculture (min. 4 mL of each monoculture, recommended 19 mL) We also need 26 mL of buffer (ddH2O, PBS, or carbon-free medium), recommended 41 mL. In what follows, we will assume that the recommended starting volumes are available. If lower volumes are available, the third step of the protocol will need to be adjusted accordingly. STEP 1: compensatory buffer to homogenize final densities -------------------------------------------------------------------------------- Label 3 96-well plates as plate_1 to plate_3 Pipette the indicated volumes of buffer into the indicated columns: volume_uL plate column 125 plate_1 1 100 plate_1 2 100 plate_1 3 100 plate_1 5 100 plate_1 9 100 plate_2 5 75 plate_1 4 75 plate_1 6 75 plate_1 7 75 plate_1 10 75 plate_1 11 75 plate_2 1 75 plate_2 6 75 plate_2 7 75 plate_2 9 75 plate_3 1 50 plate_1 8 50 plate_1 12 50 plate_2 2 50 plate_2 3 50 plate_2 8 50 plate_2 10 50 plate_2 11 50 plate_3 2 50 plate_3 3 50 plate_3 5 25 plate_2 4 25 plate_2 12 25 plate_3 4 25 plate_3 6 25 plate_3 7 STEP 2: inoculate monocultures -------------------------------------------------------------------------------- Pipette the indicated volumes of species 4 to 8 monocultures into the indicated columns: SPECIES 4 volume_uL plate column 25 plate_1 2 25 plate_1 4 25 plate_1 6 25 plate_1 8 25 plate_1 10 25 plate_1 12 25 plate_2 2 25 plate_2 4 25 plate_2 6 25 plate_2 8 25 plate_2 10 25 plate_2 12 25 plate_3 2 25 plate_3 4 25 plate_3 6 25 plate_3 8 SPECIES 5 volume_uL plate column 25 plate_1 3 25 plate_1 4 25 plate_1 7 25 plate_1 8 25 plate_1 11 25 plate_1 12 25 plate_2 3 25 plate_2 4 25 plate_2 7 25 plate_2 8 25 plate_2 11 25 plate_2 12 25 plate_3 3 25 plate_3 4 25 plate_3 7 25 plate_3 8 SPECIES 6 volume_uL plate column 25 plate_1 5 25 plate_1 6 25 plate_1 7 25 plate_1 8 25 plate_2 1 25 plate_2 2 25 plate_2 3 25 plate_2 4 25 plate_2 9 25 plate_2 10 25 plate_2 11 25 plate_2 12 25 plate_3 5 25 plate_3 6 25 plate_3 7 25 plate_3 8 SPECIES 7 volume_uL plate column 25 plate_1 9 25 plate_1 10 25 plate_1 11 25 plate_1 12 25 plate_2 1 25 plate_2 2 25 plate_2 3 25 plate_2 4 25 plate_3 1 25 plate_3 2 25 plate_3 3 25 plate_3 4 25 plate_3 5 25 plate_3 6 25 plate_3 7 25 plate_3 8 SPECIES 8 volume_uL plate column 25 plate_2 5 25 plate_2 6 25 plate_2 7 25 plate_2 8 25 plate_2 9 25 plate_2 10 25 plate_2 11 25 plate_2 12 25 plate_3 1 25 plate_3 2 25 plate_3 3 25 plate_3 4 25 plate_3 5 25 plate_3 6 25 plate_3 7 25 plate_3 8 STEP 3: 3-species landscape -------------------------------------------------------------------------------- Label 8 Falcon tubes as tube_A to tube_H Pipette the indicated volumes of buffer (ddH2O, PBS, or carbon-free medium) into the indicated tubes: volume_uL tube 9000 tube_A 6000 tube_B 6000 tube_C 3000 tube_D 6000 tube_E 3000 tube_F 3000 tube_G 0 tube_H Pipette the indicated volumes of species 1 to 3 monocultures into the indicated tubes: SPECIES 1 volume_uL tube 3000 tube_B 3000 tube_D 3000 tube_F 3000 tube_H SPECIES 2 volume_uL tube 3000 tube_C 3000 tube_D 3000 tube_G 3000 tube_H SPECIES 3 volume_uL tube 3000 tube_E 3000 tube_F 3000 tube_G 3000 tube_H Gently shake tubes to homogenize, then pipette: 75 uL per well from tube 1 into row A of plates 1 to 3 75 uL per well from tube 2 into row B of plates 1 to 3 75 uL per well from tube 3 into row C of plates 1 to 3 75 uL per well from tube 4 into row D of plates 1 to 3 75 uL per well from tube 5 into row E of plates 1 to 3 75 uL per well from tube 6 into row F of plates 1 to 3 75 uL per well from tube 7 into row G of plates 1 to 3 75 uL per well from tube 8 into row H of plates 1 to 3 FINAL LAYOUT -------------------------------------------------------------------------------- Samples are located in the positions indicated below: PLATE 1 1 2 3 4 5 6 7 8 9 10 11 12 A 00000000 00001000 00010000 00011000 00100000 00101000 00110000 00111000 01000000 01001000 01010000 01011000 B 00000001 00001001 00010001 00011001 00100001 00101001 00110001 00111001 01000001 01001001 01010001 01011001 C 00000010 00001010 00010010 00011010 00100010 00101010 00110010 00111010 01000010 01001010 01010010 01011010 D 00000011 00001011 00010011 00011011 00100011 00101011 00110011 00111011 01000011 01001011 01010011 01011011 E 00000100 00001100 00010100 00011100 00100100 00101100 00110100 00111100 01000100 01001100 01010100 01011100 F 00000101 00001101 00010101 00011101 00100101 00101101 00110101 00111101 01000101 01001101 01010101 01011101 G 00000110 00001110 00010110 00011110 00100110 00101110 00110110 00111110 01000110 01001110 01010110 01011110 H 00000111 00001111 00010111 00011111 00100111 00101111 00110111 00111111 01000111 01001111 01010111 01011111 PLATE 2 1 2 3 4 5 6 7 8 9 10 11 12 A 01100000 01101000 01110000 01111000 10000000 10001000 10010000 10011000 10100000 10101000 10110000 10111000 B 01100001 01101001 01110001 01111001 10000001 10001001 10010001 10011001 10100001 10101001 10110001 10111001 C 01100010 01101010 01110010 01111010 10000010 10001010 10010010 10011010 10100010 10101010 10110010 10111010 D 01100011 01101011 01110011 01111011 10000011 10001011 10010011 10011011 10100011 10101011 10110011 10111011 E 01100100 01101100 01110100 01111100 10000100 10001100 10010100 10011100 10100100 10101100 10110100 10111100 F 01100101 01101101 01110101 01111101 10000101 10001101 10010101 10011101 10100101 10101101 10110101 10111101 G 01100110 01101110 01110110 01111110 10000110 10001110 10010110 10011110 10100110 10101110 10110110 10111110 H 01100111 01101111 01110111 01111111 10000111 10001111 10010111 10011111 10100111 10101111 10110111 10111111 PLATE 3 1 2 3 4 5 6 7 8 9 10 11 12 A 11000000 11001000 11010000 11011000 11100000 11101000 11110000 11111000 (empty) (empty) (empty) (empty) B 11000001 11001001 11010001 11011001 11100001 11101001 11110001 11111001 (empty) (empty) (empty) (empty) C 11000010 11001010 11010010 11011010 11100010 11101010 11110010 11111010 (empty) (empty) (empty) (empty) D 11000011 11001011 11010011 11011011 11100011 11101011 11110011 11111011 (empty) (empty) (empty) (empty) E 11000100 11001100 11010100 11011100 11100100 11101100 11110100 11111100 (empty) (empty) (empty) (empty) F 11000101 11001101 11010101 11011101 11100101 11101101 11110101 11111101 (empty) (empty) (empty) (empty) G 11000110 11001110 11010110 11011110 11100110 11101110 11110110 11111110 (empty) (empty) (empty) (empty) H 11000111 11001111 11010111 11011111 11100111 11101111 11110111 11111111 (empty) (empty) (empty) (empty)