Optimized Preparation of Microalgae Chromosomal DNA

Genomic DNA agarose plugs were generated based on the protocol of Hage and Houseley [71 (link)]. Briefly, P. SENEW3 cells were cultured in f/2 medium to mid exponential growth phase cell density of 2×10⁷ cells ml⁻¹. Ten ml cell-culture aliquots were centrifuged at 3200 g for 15 mins, cell pellets were washed in 1 ml of wash buffer (10 mM Tris pH 7.6, 50 mM ethylenediaminetetraacetic acid [EDTA]) and resuspended in 50 µl of wash buffer with 1 mg ml⁻¹ lyticase. Cells were heated to 55 °C for 5 mins and mixed with 55 µl of melted 1.6 % SeaKem LE agarose in dH₂O at 55 °C and set in PFGE combe wells at 4 °C for 1 h. Solid plugs were transferred to 1.5 ml microcentrifuge tubes, digested in 500 µl wash buffer with 1 mg ml⁻¹ lyticase for 3 h at 37 °C and then in 500 µl PK buffer (100 mM EDTA, 0.2 % sodium deoxycholate, 1 % sodium lauryl sarcosine) with 1 mg ml⁻¹ proteinase K (Sigma-Aldrich P6556) overnight at 55 °C. Digested cell plugs were washed three times in 1 ml wash buffer for 30 mins at room temperature, resuspended in 500 µl wash buffer and stored at 4 °C.
P. SENEW3 chromosomal DNA was separated using a Bio-Rad CHEF Mapper XA PFGE system. Four agarose plugs and a NEB Yeast Chromosome PFGE marker (N0345S) plug were inserted into a 1 % SeaPlaque Low Melting Point Agarose (Lonza: 50101–25 g) gel, prepared with 0.5 X Tris/Borate/EDTA (TBE) buffer. Each plug was run in parallel for 24, 28, 32 and 36 h, respectively (Table S1, available in the online version of this article). Following each time point, each lane was excised and visualized with a blue light gel box.