DNA probes were prepared from unmodified and chromophore-labeled DNA oligonucleotides synthesized by Integrated DNA Technologies. Target double-stranded (ds) DNA and Cas9 beacon DNA constructs shown in Figure 1A were formed by mixing equimolar amounts of synthetic complementary strands (final concentrations were within low μM range) in a buffer containing 40 mM Tris, pH 7.9, 100 mM NaCl; heating for 2 min at 90°C and slowly cooling the reactions to 20°C. The protospacer adjacent motif (PAM)-distal ends of the beacon target and non-target strands are labeled with fluorescein and Iowa Black® FQ, respectively. pUC19 plasmid vector was purchased from New England Biolabs.
Puc19 plasmid vector
Manufactured by New England Biolabs
PUC19 is a commonly used plasmid vector. It is a small, circular DNA molecule that can be used for cloning and propagation of DNA sequences in Escherichia coli (E. coli) bacteria. The PUC19 vector contains an antibiotic resistance gene, which allows for selection of bacterial cells that have successfully taken up the plasmid.
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Lab products found in correlation
2 protocols using puc19 plasmid vector
1
Fluorescent DNA Probe Preparation
2
Construction of SIAH-Binding Deficient HSV-2 ICP0 Mutants
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To construct the SIAH-binding deficient HSV-2 ICP0 mutants, transfer plasmids were generated. First, a homology plasmid was constructed by subcloning two DNA fragments derived from wild-type HSV-2 MS genomic DNA into a pUC19 plasmid vector (New England Biolabs), encompassing sequences homologous to 517 bp upstream of the HSV-2 ICP0 start codon and 545 bp downstream of the ICP0 stop codon. During cloning, XbaI and BamHI restriction sites were introduced in-between the two homology arms. Next, pICP0-GFP plasmids (see above) and the homology plasmid were treated with the enzymes XbaI and BamHI and fragments were separated via gel electrophoresis. The linearized homology plasmid as well as the XbaI-BamHI DNA fragment (ICP0-GFP sequence) from the pICP0-GFP plasmids, were extracted and the ICP0-GFP sequence was ligated into the homology plasmid using the DNA Rapid Dephosphorylation and Ligation Kit (Roche). The resulting transfer plasmids, where the wild-type or mutated ICP0-GFP sequence is flanked by the homology arms, are shown in S1 Table .
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