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 .
