A 2038 nt ssDNA was generated by Guide-it™ Long ssDNA Production System (Takara) using oligo 11 and oligo 12 as primers. The exonuclease activities were examined using 15 nM of the 2038 nt ssDNA substrate incubated with 20 nM exonuclease (for EcExoI or EcRecJ) or 0.33 U/μL (for EcExoVII) in 30 μL reaction containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 125 mM NaCl and 0.1 mM DTT at indicated temperature for 1–30 min. Where indicated, 2 μM EcSSB (Thermo Fisher Scientific) or 500 nM EcUvrD plus 1 mM ATP were included in the exonuclease assay. The reactions were stopped by addition of 25 mM EDTA, 0.2% SDS, 0.67 mg/mL Proteinase K (Denville) and incubated at 50 °C for 1 h. Resulting DNA was resolved on a 1% agarose gel, scanned on a Sapphire Biomolecular Imager (Azure Biosystems) and quantified by ImageQuant software.
Guide it long ssdna production system
Manufactured by Takara Bio
Sourced in United States
The Guide-it™ Long ssDNA Production System is a laboratory equipment designed to produce long single-stranded DNA (ssDNA) fragments. It is a tool used in various molecular biology and genomic research applications.
Automatically generated - may contain errors
Lab products found in correlation
Sapphire biomolecular imager, by Azure Biosystems (1 mentions)
Gibson assembly cloning kit, by New England Biolabs (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 plasmid, by Thermo Fisher Scientific (1 mentions)
In fusion hd cloning plus kit, by Takara Bio (1 mentions)
Nucleospin gel and pcr clean up kit, by Takara Bio (1 mentions)
Nucleobond xtra midi ef, by Macherey-Nagel (1 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
8 protocols using guide it long ssdna production system
1
Exonuclease Activity Assay Protocol
2
Precise PA-tag Integration in Mice
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To insert three tandem copies of PA-tag sequences just upstream from the stop codon (Fig. 1b ), 3×PA-tag sequences flanked by 129- and 118-bp homology arms were artificially synthesized (Integrated DNA Technologies). To avoid unwanted excision of PA-tag sequences by the internal recombination among three copies, synonymous substitutions were introduced into each tag sequences. By using this synthesized DNA as the template, long single-stranded DNA (ssDNA) donors were prepared with the Phospho-PCR method (Guide-it Long ssDNA Production System, Takara Bio; Inoue et al., 2021 (link)). The mixture of annealed crRNA/tracrRNA, Cas9 protein, and long ssDNA donor was injected into mouse zygotes at the concentrations of 38, 50, and 25 ng/μl, and the zygotes were transferred into the oviduct of surrogate mothers to raise pups.
3
Single-stranded DNA production for HDR
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To produce long ssDNA as HDR templates, the DNA of interest was amplified via PCR using one regular, non-modified PCR primer and a second phosphorylated PCR primer. The DNA strand that will be amplified using the phosphorylated primer, will be the strand that will be degraded using this method. This makes it possible to prepare either a single-stranded sense or single-stranded antisense DNA using the respective phosphorylated PCR primer. To produce the ssDNA strand of interest, the phosphorylated strand of the PCR product was degraded by treatment with two enzymes, Strandase Mix A and Strandase Mix B, for 5 minutes (per 1kb) at 37C, respectively. Enzymes were deactivated by a 5 minute incubation at 80C. The resulting ssDNA HDR templates were SPRI purified (1.0X) and eluted in H2O. A more detailed protocol for the Guide-it™ Long ssDNA Production System (Takara Bio USA, Inc. #632644) can be found at the manufacturer’s website.
4
Easi-CRISPR Knock-in Protocol
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We amplified two PCR products: 1811 bp upstream (fragment A) and 2001 bps downstream (fragment C) of the cutting site from the selected sgRNA (#3). Fragment B containing floxed-miniSTOP (393 bps) was amplified from a plasmid that was synthesized by GenScript. The floxed-miniSTOP sequence is shown in Supplementary Materials . Fragments A, B, and C were combined using the Gibson Assembly Cloning Kit (#E510, NEB), and then ssODN was generated using Guide-it™ Long ssDNA Production System (#632644, TaKaRa Bio USA, Mountain View, CA, USA). Three hundred and ninety-three bases of the floxed-miniSTOP cassette and homologous sequences at both ends (115 bases and 145 bases) corresponded to the cutting site of sgRNA#3 (Figure 2 b, right). The ss-ODN sequence is shown in Supplementary Materials . Easi-CRISPR was performed using the conventional pronuclear injection by Dr. Chyuan-Shen Lin, as previously described [44 (link)].
5
Targeted GFP Integration by ssDNA
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ssDNA insert was designed to consist of GFP driven by CMV promoter, flanked by two 400 bp regions homologous to IgH and c-myc regions near to breakpoint. IgH and c-myc homology regions were added in order to facilitate specific integration of GFP + CMV insert into reciprocal translocation breakpoint. ssDNA insert was generated using Guide-it Long ssDNA production system (Takara Bio). Briefly, we assembled 400 bp homology regions (ordered as GeneBlocks from IDT), GFP + CMV insert (amplified by PCR from pmax-GFP plasmid (Amaxa, Lonza) using GFP-PCR1 primers) and backbone from pcDNA3.1 plasmid (Invitrogen, Thermo Scientific) using In-Fusion HD Cloning Plus kit (Takara Bio). Then, we generated PCR product using Phusion High-Fidelity DNA polymerase (Thermo Scientific) and STR1 primers and digested sense strand of PCR using Strandase enzyme (from ssDNA production system). ssDNA was then isolated using NucleoSpin Gel and PCR Clean-Up Kit (Takara Bio).
6
Floxed Exon 4 Knock-in Vector Construction
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To construct the floxed exon 4 knock-in vector, three amplified PCR products containing loxP sequences were cloned into the XhoI–NotI site of pBRBlue II, then two amplified PCR products containing two substituted bases from the vector were cloned into the XhoI–NotI site of pBRBlue II. An amplified PCR product from the vector was used as the template for the long ssODN, which was produced using the Guide-it Long ssDNA Production System (TaKaRa). The primers for PCR are listed in Supplementary Table 2 .
7
Plasmid Construction and Donor Preparation
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Plasmid constructs were prepared with NucleoBond Xtra Midi EF (Macherey-Nagel) according to the manufacturer’s protocol and Sanger sequence confirmed (Eurofins Genomics, Ebersberg, Germany). The antisense (also the strand that sgRNA-190 binds to) ssDNA (primers: 5’-phosphorylated LA587 and LA588) and BTN-ssDNA (primers: 5’-phosphorylated LA587 and 5’-biotinylated LA588) donors of 190-recoded were generated using the Guide-it Long ssDNA Production System (Takara Bio). Biotinylated donors were amplified from their respective plasmid backbones with 5’-biotinylated primers LA2967 and LA2968. All non-plasmid donors were gel extracted and purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) and ethanol precipitated before injection mix preparation.
8
Single-stranded DNA production for HDR
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