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The donor DNA, which includes the sequence encoding a fluorescent protein, is provided to the cell to repair a double-strand break induced by a nuclease.
The ratio of the concentration of FAIRE DNA (which includes only open chromatin regions) to that of the FAIRE control DNA sample (whole genomic DNA) was used as the threshold.
Previous research had found that a type of DNA which is passed down only by mothers, known as mitochondrial DNA, is more similar between Bornean and Tapanuli orangutans, but nuclear DNA – which includes genes from both parents – is more similar between Sumatran and Tapanuli orangutans.
DR- white was created using a three-way Gateway LR Clonase II reaction of Sce.white, 5.1 kb of chromosome X DNA (which includes the y + transgene), and iwhite.attB (Life Technologies, Grand Island, NY) following the manufacturer's protocol.
The resulting plasmid fused 2.1 kb of 5' flanking DNA, which includes the last intron and exon of the adjacent upstream feh-1 gene, and the first three predicted exons of the nhr-239 gene to the coding sequence for GFP.
Addition of PcrA to a preformed complex of RepD and Junction 1 DNA, which includes the ICRIII duplex sequence, causes a further increase in anisotropy, indicating that PcrA is binding to the RepD-DNA complex with a Kd of 22 nM.
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We designed primers to amplify genomic DNA, which included at least 300 bp of the terminal exon.
This description would fit with the three dimensional descriptions of DNA which include loop attachments [ 40, 41].
A total of 5 × 10 cells were transfected with 5 10 μg total plasmid DNA, which included the firefly luciferase-expressing vector using the DEAE-dextran procedure.
We sequenced five nuclear loci for a subset of our samples that contained high quality DNA, which included 18 Aneides lugubris (representing 15 localities) and three outgroups (Aneides flavipunctatus-MVZ19969, A. vagrans-MVZ269484, and A.ferreus-MVZ269393).
On Day 1, we amplified the biotin-conjugated spacer DNA, which included I- SceI and SfiI recognition sequences from the pUC19-I SceI plasmid and was used for all three experiments; this DNA was digested with SfiI and attached to streptavidin-coated beads as the initial acceptor DNA.
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