Notebook: P. tricornutum Next

Phaeo Quick Start Guide


The cultivation of P. tricornutum requires continuous light (8000-11000 lux) at 22° C. Liquid cultures are constantly shaken at 225 rpm in Erlenmeyer flasks containing f/2 medium. Use parafilm for f/2 agar plates to avoid dehydration. For strain maintenance use selective plates and transfer the cells to new plates every four to six weeks.


  • Microcarrier preperation

    Transfer 60 mg Tungsten M 10 Microcarrier into a sterile 1.5 Eppendorf cup and add 1 ml sterile EtOH abs. Vortex the suspension 1 min and centrifuge for 1 min with 20000x g at room temperature. Discard the supernant and resuspend the pellet in 1 ml sterile ddH2O, thereafter centrifuge (1 min/20000 g/RT). Repeat this step and finally resuspend the pellet in 1 ml of sterile ddH2O. Divide the suspension into 50 µl aliquots. You can store them at -20 °C. ! Vortex the final 1 ml suspension after every aliquot to avoid sedimentation.

  • Preperation of P. tricornutum

    You need 100 µl of a 108 cells containing suspension for a single transfection. About one week after inoculation you assess the cell number of your liquid culture by a counting chamber. We used 5 µl of the liquid culture to load a Thoma counting chamber. With the resulting average cell number per small squares, the total cell number were calculated as follows: Cell number= Ø cells/small square * 4 (chamber factor) * 106 * volume of liquid culture (ml) You pelletize the calculated volume by centrifugation (5 min/ 1500 g/RT) and discard the supernant afterwards. Resuspend the pellet in an appropriate volume and transfer 100 µl to the middle of an f/2 agar plate. Use a sterile inoculation loop to spread out the suspension from the center to half the radius of the plate. Incubate the cells for 12-24 hours at conditions given above before you use them for transfection.

  • DNA precipitation to the microcarriers

    Transfer 5 µg DNA, 50 µl CaCl2 and 20 µl Spermidine (0.1 M) into the 50 µl Tungsten-aliquot tube and vortex the mixture 1 min. After 10 min of sedimentation discard the supernant and resuspend the pellet with 250 µl EtOH (HPLC grade). Repeat the vortex-sedimentation-steps. Discard the supernant and resuspent the pellet with 50 µl EtOH (HPLC grade). Now you can use the particles for transfection. The prepareted particles are sufficient for three transfection. ! We recommend to use DNA received by midi-preperation with a minimum concentration of 1000 ng/µl. DNA received by mini-preparation leads to drastical reduction of transfection efficiency.

  • Biolistic transfection

    We achieved the transfection by using the Biolistic PDS-1000/He Particle Delivery System from Biorad. Before you can start the transfection procedure, perform a cleaning shot without DNA. Thereafter clean the gun and every single unit you need for transfection with EtOH abs. (HPLC grade). After all ethanol evaporated transfer 15 µl of the microcarriers onto the macrocarriers lying on the macrocarrier holders. Assemble the components of the gun by following the producer’s manual. Induce the shot with a vacuum of -25 psi and a pressure of 1350 psi. Release the vacuum immediately after the ripping-event of the rapture-disc. After the bombardment, seal the plates with Parafilm and incubate at 22 °C under continuous light for 24 hours.

  • Wash step

    5. Wash step After the incubation use 1 ml f/2-medium to wash the cells from the plates. Spread the cell suspension onto three f/2-zeocin-agar plates equally and thereafter seal the plates with parafilm. The following period of incubation occurs at the conditions given above until the arising colonies are big enough for further analysis (Ø < 1 mm). Then pick the colonies to new selective plates.

Confocal laser scanning microscopy

For the constructs tagged with GFP we used a Leica TCS SP2 laser scanning microscope (Leica) for the workable confirmation of our biobricks. The protein expression was induced with light or nitrate according to the used promotor.

Material required for biolistic transfection

  • Biolistic PDS-1000/He Particle Delivery System (Bio-Rad)
  • 1350 psi Rapture Discs (Bio-Rad)
  • Macrocarrier (Bio-Rad)
  • Macrocarrier Holders (Bio-Rad)
  • Stopping Screens (Bio-Rad)
  • Tungsten M 10 (Ø 0.7µm) Microcarrier (Bio-Rad)


Tropic marine sea salt   1,6 % (w/v)
Tris/HCL pH 8   2 mM
NaNO3 or rather NH4Cl   0.9 mM or rather 1.5 mM
NaH2PO4   36 μM

F/2-micronutritions solution

FeCl3   11,65 mM
Na2EDTA   11.71 mM
CuSO4   39 μM
ZnSO4   77 μM
CoCl2   42 μMM
Na2MoO4   26 μM

Solution added to the medium before usage with a dilution of 1:1000.

F/2-vitamin solution

Biotin   2 μM
Cyanocobalmin   0.37 μM
Thiamin-HCl   297 μM

Solution added to the medium before usage with a dilution of 1:1000.

  • For f/2 solid medium: add 1.5 % (w/v) agar
  • For selection medium: add 75 µg/ml zeocin

Phaeo Notebook

Investigator: Marian

315   pSB1A3 486476315 (antibody construct)
496   pSB1A3 496315 (NR promoter)
416   pSB1A3 395416 (FcpB promoter)
410/411   pSB1A3 4+12/13+96315 (stroma localization of eGFP)
412/413   pSB1A3 4+10/11+96315 (ER localization of eGFP)


Transfection of 315 and 416 according to protocol (see above).


Wash cells (27.07.13) according to protocol (see above).


Transfection of 496 and 315 according to protocol (see above).


Wash cells (08.08.13) according to protocol (see above).


Received colonies of 416: Transfer to fresh plate.


Transfection of 315, 410, 411, 412, 413, 496 according to protocol (see above).


Wash cells (28.08.13) according to protocol (see above).


Inoculation of seven 315 colonies into liquid medium.


Inoculation of 18 416 clones into liquid medium.

Transfer of 496, 411, 412, 413 to fresh plates.

410: no colonies.


Transfer 496 to nitrate-plates for induction.


Analyze 496 and 416 at CLSM.

The results demostrate that both promotors (i.e. PNR and PfcpB) work properly as shown by the expression of eGFP (green, second panel). The red color (third panel) is due to autoflourescence of the plastides and indicates their localisation within P. tricornutum. The very left and the very right panel represent DIC and an overlay of green and red, respectively.