TITLE: CRISPR-Cas9 mutagenesis in Phaeodactylum tricornutum, particle bombardment
AUTHORS: Mark Moosburner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Particle bombardment mediated transformation of CRISPR-Cas9 components to the diatom Phaeodactylum tricornutum</div></div>

[STEPS]
?. [Phaeodactylum cell preparation]
Plate wild-type Phaeodactylum in preparation for particle bombardment
?. [Phaeodactylum cell preparation]
Calculate the cell concentration of a culture of Phaeodactylum tricornutum that is grown axenically. Calculate the volume required for 1x108 cells total. Pellet at 2000 x g for 20 minutes at 17oC.
?. [Phaeodactylum cell preparation]
Dry growth agar plate under a PCR hood while cells are pelleting    Agar Plate = 15mL 1.6% agarose + 15mL NO3-ASW (artificial sea water)
?. [Phaeodactylum cell preparation]
Remove media from pellet and resuspend in 200uL ASWSpread on a plate evenlyAllow cells to completely dry on plate while under PCR hoodSeal plate w/ breath-easy tape
?. [Phaeodactylum cell preparation]
Grow cells on plate for 4 days
?. [Cas9 and sgRNA plasmid preparations]
Prepare plasmids for particle bombardment *** start 2 days prior to bombardment ***
?. [Cas9 and sgRNA plasmid preparations]
2 days  prior to bombardment....Obtain sequence verified expression plasmids for Cas9 and sgRNA from frozen bacterial stocks.Grow up 15 mL of each plasmid OVN
?. [Cas9 and sgRNA plasmid preparations]
Extract and purify all plasmid the next day by performing 3 5mL mini-preps for each 15mL culture.*** at the end of all mini-preps, elute the 3 columns pertaining to one plasmid into a single collection epitube ***
?. [Cas9 and sgRNA plasmid preparations]
Measure the plasmid concentrationCalculate the volume required for 8-ug plasmid DNA for each plasmid
?. [Particle Bombardment]
Prepare plasmids
?. [Particle Bombardment]
Pipette 8ug of each plasmid into one 1.5mL epitube.Use a speed-vac to concentrate tdNA to a total of 5-10uL
?. [Particle Bombardment]
Prepare particle bombardment materials- Turn on helium tank to pressure = 200psi- Obtain vacuum pump and set up with PDS-1000- Re-suspend tungsten beads in 50% glycerol (vortex to mix)- Obtain a plat-form vortex- Prepare 2.5M CaCl2- Prepare 0.1M Spermidine- Prepare 70% EtOH- Obtain 100% EtOH- Obtain Isopropanol- 1 rupture disk- 7 macrocarrier disks- Place Phaeodactylum plate in PCR with the PDS-1000 system to allow the plate to dry before bombardment
?. [Particle Bombardment]
Mix tungsten bead by vortexPipette 50uL beads into 1.5 epitube.Platform vortex for 5 minutesDuring the 5 minutes, sterilize the 7 macrocarrier disks.    - pour 70% EtOH into a petri dish   - wash each disk using tweezers by shaking the disk in the 70% EtOH   - dry on other half of petri dish
?. [Particle Bombardment]
After 5 min on plate vortex...Pipette DNA into tungsten beachPipette 50uL CaCl2 (2.5M), vortexPipette 20uL Spermidine (0.1M) to hybridize DNA to beadsMix by flicking tube with finger MANY TIMES. Plate vortex for 3 minutes
?. [Particle Bombardment]
During 3 minutes....Place 7 dried macrocarrier disks into the carrier disk manifold. Ensure disks are secured under the lip of each macrocarrier position.
?. [Particle Bombardment]
After 3 minutes....Pellet tungsten beads in a table-top centrifuge (fixed rotor) Centrifuge at 8000 rpm for 10 seconds (beads will pellet very easily)Remove supernatentResuspend in 140uL 100% EtOH, MIX THOROUGHLY by pipetteRepeat pelleting and resuspension 2XOn last resuspension, add only 100uL 100% EtOH and MIX THOROUGHLYMAINTAIN HOMOGENY BY FLICKING TUBE
?. [Particle Bombardment]
Pipette 10uL of beads onto each macrocarrier diskAllow beads to dry completely on disks
?. [Particle Bombardment]
While drying....Briefly wash rupture disk in isopropanolInsert "wet" rupture disk into rupture manifold and screw manifold into the bombardment systemSecure the metal mesh screen between the two plates of the carrier disk manifold.Insert manifold into square metal fitting and insert into the appropriate slot of the bombardment system.(The rupture manifold should be suspended directly above the carrier disk manifold and aligned with the 7 holes)
?. [Particle Bombardment]
Place the Phaeodactylum plate below and in line with the carrier disk manifold
?. [Particle Bombardment]
Shut door to the bombardment systemTurn on the vacuum manifold. Allow the vacuum pressure to build to 25 inch Hg and hold.Press and hold the "FIRE" button. The pressure will build until ~1300 psi when the rupture disk ruptures. There will be a loud bang! Once the loud bang occurs, "VENT" the bombardment system to remove the vacuum pressure.
?. [Particle Bombardment]
Place the lid on the Phaeodactylum plateWrap the plate in tin foilIncubate the plate at 18oC for 48hrs
?. [Select for transformed Phaeodactylum cells]
After 48hrs in the dark...Scrape cells off plate and resuspend in a 2mL epitube with ASW.
?. [Select for transformed Phaeodactylum cells]
Prepare selection platesEach plate: 15mL 1.6% agarose + 15mL ASW (w/ appropriate N-substrate) + Phleomycin antibiotic (20mg/mL)Dry plates under PCR hood before plating cells
?. [Select for transformed Phaeodactylum cells]
For each transformation, dry 3 selection plates.Plate all cells on the 3 plates and spread (maximum 400uL per plate)Allow cells to completely dry under PCR hood. *** Phleomycin resistant colonies appear after 3-4 weeks***