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1. #Neenah creative collection starter pack target verification
2. #Neenah creative collection starter pack target download

#Neenah creative collection starter pack target download

Download FIG S5, TIF file, 0.2 MB.Īgarose gel electrophoresis assays for nuclease activity in components and steps of the genetic transformation procedure. The vector sequences adjacent to the 5′ and the 3′ genomic DNA target site were preserved, but most of the intervening vector sequences were absent, likely due to deletion mediated by homologous recombination between sequence repeats during meiosis. The insert sequence amplicon from the UF1- oah1-3-6-② meiotic progeny was fully sequenced by Sanger sequencing primer walking and verified by PCR amplification (C). This is likely due to the insertion of multiple duplicated sequences. Thus, the vector DNA insertion in UF1- oah1-3-6 was much larger and more complex than we could ascertain from Illumina genome sequence assembly and PCR. PCR amplification results with the F1, F2, F3, and F4 primers paired with Fsg3 as the reverse primer produced expected ~4- and ~5-kb amplicons for F4×Fsg3 and F2×Fsg3, respectively, but a much smaller, ~1.5-kb, amplicon for F1×Fsg3 indicative of the presence of a second copy of the sequence encoding the F1 primer (C). These results indicate the presence of large (>10-kb) intervening sequences, including a second copy of the F4 primer on the opposite strand of the inserted DNA (B). Using vector primers F1, F2, F3, and F4 with the 3′ genomic DNA R primer produced only an amplicon with the F4×R combination however, when the F4 primer was used alone, an amplicon of the same size, ~9.5 kb, was produced. Additional PCR results using vector-vector and vector-genomic DNA primers indicated that the actual insertion was larger than the 11-kb vector sequence (B and C). Likewise, amplification with flanking genomic DNA primer “R” and vector sequence Rsg3 validated the 3′ insertion junction from UF1- oah1-3-6 (A). Amplification using flanking genomic DNA primer “F” and vector primers “R2” and “R3” ( Fig. 3B) validated the 5′ insertion junction of UF1- oah1-3-6 obtained from TAIL-PCR ( Fig. 1) and the genome assembly (A).

To test the validity of the insertion sequence structure depicted in Fig. 3B, PCR amplifications were performed within the insertion and across the insert-genomic DNA junction in both the primary transformant, UF1- oah1-3-6, and its meiotic progeny, UF1- oah1-3-6-②.

#Neenah creative collection starter pack target verification

PCR verification of the UF1- oah1-3-6 and UF1-3-6-② CRISPR-Cas9-inserted sequences.