Fig. 2: Comparing the transformation efficiency of linear, circularized, and phosphorylated DNA.

a An agarose gel depicting different preparations of the episome pPtGE31_ ΔPtR. CP: circular plasmid, EcoRI: restriction enzyme digested plasmid, EcoRI^MBN: restriction enzyme digested plasmid with additional mung bean nuclease treatment to remove sticky ends, PCR: PCR-amplified linear plasmid DNA, and PCR^phos: PCR-amplified linear plasmid DNA generated with phosphorylated primers. b Comparison of P. tricornutum transformants electroporated with different preparations of pPtGE31_ ΔPtR. Following electroporation, one-tenth of the total reaction was plated on ¼-salt L1 plates supplemented with 100 µg/ml nourseothricin. Plates are labeled according to the type of DNA that was transformed, as described above. The number of CFUs is shown in the bottom right corner of each plate. c A plasmid map depicting the regions that were screened when assessing passaged algal transformants. The 486 bp junction spans the region where pPtGE31_ ΔPtR was split for PCR amplification, whereas the 334 bp junction spans an intact region of the plasmid backbone. d Screening 10 algal colonies that had been transformed with circular pPtGE31_ ΔPtR or e PCR-amplified pPtGE31_ ΔPtR. The primer pairs span the termini region (expected size of 463 bp, top panel) and the CEN/ARS region of the episome backbone (expected size of 334 bp, bottom panel). Primer pairs are listed in Supplementary Data 2. Dilute pPtGE31_ ΔPtR and genomic P. tricornutum DNA were used for the positive and negative controls, respectively. Double asterisks (**) indicate the algal episomes that would be recovered in E. coli and sent for whole plasmid sequencing.