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It differs because it is only compatible with vectors that contain UCSs, but the superior performance compared to the more versatile In-Fusion system makes USER cloning a more attractive technique for large-scale gene replacement projects. Four binary vectors with UCSs and the hygromycin B selection marker gene are available for use in ascomycetes (24). New selection marker cassettes can easily be introduced into these vectors, and the series have been extended to include vectors with geneticin/G418 (nptII gene) and glufosinate ammonium (bar gene) resistance markers (data not published).
Random integration into the genome via non-homologous end joining (NHEJ). 5. Integration by HR via a single crossover reaction between upstream or downstream HRSs in circular T-DNA and the target locus (scHR). 6. Or combinations of the above. Complete degradation of the T-DNA or extensive truncation of the T-DNA ends results in the loss of the selection marker gene 2 Targeted Gene Replacement in Fungal Pathogens¼ 25 and the recipient cell will not survive the imposed selection regime. The formation of circular T-DNA molecules typically leads to unstable replication due to a lack of an autosomal replication sequence in the T-DNA and will eventually result in loss of the selection marker.
Using a pipette, place 1 mL of plasmid DNA on the internal side of the cuvette. 4. Use 50 mL of competent cells to gently flush the DNA containing droplet to the bottom of the cuvette. Gently thump the cuvette on a table a couple of times to remove air bubbles. Wipe down the exterior sides of the cuvette with a paper towel (to prevent short circuits). 5. 50 kV, Capacitance = 25 mF, Resistance = 200 W) and shock. The accumulator should discharge the stored energy in approximately 5 m s (see Note 14).