Pulse Field Gel Electrophoresis (PFGE) Protocol for separation of Chlorella chromosomal DNA and Chlorella virus DNA
Harvest Chlorella NC64A (or Pbi) cells from 4 day old cultures (1.2 - 2.0 x 107 cells/ml) by centrifugation at 4000 x g for 5 minutes.
Re-suspend in MBBM (NC64A cells) or FES (Pbi cells) at concentration of 8.6 x 107 cells/mL.
Add chlorella virus to a multiplicity of infection (MOI) = 10 plaque-forming units (pfu)/cell.
Sample infected chlorella cells (25 mL) into prepared centrifuge tubes with formaldehyde (the final formaldehyde concentration is 4%) and place on ice.
Centrifuge at 4000 x g for 5 minutes.
Wash samples by re-suspending them in 10 mL of MBBM amended with 50 mM EDTA and following centrifugation at 4000 x g for 5 minutes.
Repeat wash step 3 times.
Re-suspend washed infected cells in 0.5 mL of SB.
Add to the cells 0.5 mL of 2% low melting point agarose (BioRad) in SB (kept at 45°C), mix well (work quickly, try not to generate any air bubbles), and pour the mix into BioRad plug molds.
Place plug molds in refrigerator for 15 minutes to solidify.
Carefully remove agarose blocks from mold and place them into 2 mL of DB amended with 1mg/mL Proteinase K.
After incubation, wash agarose blocks for 30 minutes with DB 4 times.
Cut blocks in small pieces to fit gel wells.
Load agarose blocks into gel wells and seal them with melted (~45°C) 1% low melting point agarose in running buffer.
The chromosomes of Hansenula wingei (1.05-3.13 Mbp), cat#170-3667; Schizosaccharomyces (3.5-5.7 Mbp), cat#170-3633 (Bio-Rad, Hercules, CA, USA), and Yeast Chromosome PFG Marker (225-1,900 Kbp), cat#N0345S (New England Biolabs, Beverly, MA, USA) are used as molecular weight markers.
Separate chromosomal DNA  in CHEF-DR  (BioRad, Hercules, CA) electrophoresis unit with 1X TAE running buffer.
Run electrophoresis at 3 V/cm (100 V) with pulse time ramping from 250 to 900 seconds for 60 hrs.
Change buffer  every 24 hrs.
Stain gel with 0.5 mg/L ethidium bromide for 20.
Add 3.1 ml of 37% formaldehyde into 40 ml centrifuge tubes and place them on ice.
Incubate agarose blocks for 24 hrs at 50°C Prepare 1% agarose gel (PFGE grade) in 1× TAE buffer using BioRad casting stand.
