Genomic DNA Extraction and Genotyping of Dictyochloropsis Green Algae Strains

Materials and Reagents

1. Qiagen Type-it Microsatellite PCR kit (QIAGEN, catalog number: 206243 )
   
2. 1x TE buffer (10 mM Tris, bring to pH 8.0 with HCl, 1 mM EDTA)
   
3. GelRed (Biotium, catalog number: 41003 )
   
4. 1 kb DNA ladder (GeneRuler 1 kb DNA Ladder) (Thermo Fisher Scientific, catalog number: SM0313 )
   
5. Nuclease-free water
   
6. Pure molecular biology grade ethanol (96–100%)
   
7. 2 ml tubes
   
8. DNeasy 96 Plant Kit (QIAGEN, catalog number: 69181 )
   
9. Hi-Di™ formamide (Life Technologies, catalog number: 4311320 )
   
10. GeneScan 500 LIZ size standard (Life Technologies, catalog number: 4322682 )
    
11. Ice
    
12. Glucose (BD Biosciences, catalog number: 215510 )
    
13. Proteose pepton (BD Biosciences, catalog number: 212230 )
    
14. Agar (BD Biosciences, catalog number: 214883 )
    
15. 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (Life Technologies, Gibco^®, catalog number: 11330032 )
    
16. Laboratory wipes (e.g., KimWipes, Kimberly-Clark, catalog number: TW31KWPBX )
    
17. 4 mm steel balls (e.g., Spex Certiprep, catalog number: 12145950 )
    
18. Pipette tips
    
19. Pipette (0.1-2 μl), pipette tips
    
20. Processing plate: 96-well plate (e.g., MicroAmp Optical 96-Well Reaction Plate) and 96-well plate (Septa)
    
21. 0.22 µm sterile polyethersulfon syringe filter (Merck Millipore, catalog number: P/N SLMP025SS )
    
22. Algal medium (see Recipes)
    
23. Algal culture medium preparation for growing Dictyochloropsis and other trebouxiophycean algal strains (see Recipes)
    
24. Vitamin solution (see Recipes)
    

Equipment

1. Multi-channel pipettes (0.1-10, 10-100, 100-1000 μl), with extended tips
   
2. Microcentrifuge with rotor for 2 ml tubes
   
3. Centrifuge for 96-well plates
   
4. BioRad Gel casting tray, running tray, power pack etc. (Bio-Rad Laboratories, catalog number: 164-0305 )
   
5. Incubator (65 °C)
   
6. Freezer or cold room at -20 °C
   
7. PCR Thermal cycler
   
8. ABI3130/ 3130xl/3730/3730xl DNA Analyzer (Applied Biosystems)
   
9. Ball Mill MM 400 (Retsch^®, catalog number: 20.745.0001 )
   
10. Lyophilizer (e.g., FreeZone 4.5 Liter Benchtop Freeze Dry System, Labconco, catalog number: 7750021 )
    

Software

1. GeneMapper^® (Software v4.1, Applied Biosystems, catalog number: 4366925)

Procedure