Cviaii

Manufactured by New England Biolabs

Sourced in United States

CviAII is a type II restriction endonuclease that recognizes and cleaves the DNA sequence 5'-RGCY-3', where R is a purine (A or G) and Y is a pyrimidine (C or T). This enzyme is useful for DNA manipulation and analysis.

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Lab products found in correlation

8 protocols using cviaii

Telomere Length Measurement Protocol

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Telomere shortest length assay was performed as described (Lai et al., 2017). Genomic DNA was ligated to telomere linkers by T4 DNA ligase (NEB), followed by digestion with a mixture of restriction enzymes CviAII, BfaI, NdeI, and MseI (NEB). DNA was treated with Shrimp Alkaline Phosphatase (NEB) followed by adaptor ligation. 1 μM of AT and TA adapter was ligated to the DNA fragments by T4 DNA ligase. Telomeres were amplified by PCR (94°C for 2 min, 26 cycles of 94°C for 15 s, 60°C for 30 s, and 72°C for 15 min) and resolved on a 0.85% agarose gel (1.5 V/cm for 21 hr). Southern blot was performed as in TRF analysis to detect telomeres.

Li Y., Zhou G., Bruno I.G., Zhang N., Sho S., Tedone E., Lai T., Cooke J.P, & Shay J.W. (2019). Transient introduction of human telomerase mRNA improves hallmarks of progeria cells. Aging Cell, 18(4), e12979.

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CRISPR and TALEN-mediated Genome Editing of miRNAs

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As previously described, TALENs were used to create miR-139 and miR-223 mutants and a multiplexed pool of CRISPR guide RNA pairs and Cas9 mRNA was used to simultaneously mutagenize all four miR-24 genes (Narayanan et al., 2016 (link); Ristori et al., 2015 (link)). Both strategies were carried out in the Tg(kdrl:gfp)^la116 background. Mutagenesis was initially determined by restriction endonuclease assay using CviAII (NEB) for the miR-139 locus and T7 endonuclease I (NEB) for miR-24 and miR-223 genes. Briefly, 50 ng of genomic DNA isolated with the DNeasy Blood and Tissue kit (Qiagen) from a clutch of 15–20 injected 24 hpf embryos was used to amplify a 200–400 bp region spanning the intended mutation site. Differential restriction endonuclease patterns of PCR amplicons indicated successful genome editing. Once mutagenesis was confirmed, embryos remaining in the same clutch were raised. F0 founder fish were identified and outcrossed first with AB wild type and then with the Tg(kdrl:gfp)^la116 strains to allow visualization of the developing vasculature. Mutations in adult F0 founders and subsequent generations were identified by 6-FAM fluorescent PCR fragment analysis. Genotyping PCR primers are listed in Table S2. We characterized the nature of the mutant allele by cloning the mutant PCR product in a pGEM-T Easy vector (Promega) and sequencing the resulting plasmid.

Kasper D.M., Moro A., Ristori E., Narayanan A., Hill-Teran G., Fleming E., Moreno-Mateos M., Vejnar C.E., Zhang J., Lee D., Gu M., Gerstein M., Giraldez A, & Nicoli S. (2017). microRNAs Establish Uniform Traits during the Architecture of Vertebrate Embryos. Developmental cell, 40(6), 552-565.e5.

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Telomere Length Profiling with TeSLA

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The TeSLA procedure was carried out as described in Lai et al. (2017) (link), with minor modifications. Oligonucleotide sequences for the ligation and amplification reactions were published in Lai et al. (2017) (link).
Briefly, 50 ng of genomic DNA was ligated with TeSLA-T oligonucleotides and then digested with CviAII, BfaI, NdeI, and MseI restriction enzymes (New England Biolabs), followed by shrimp alkaline phosphatase (New England Biolabs) treatment. The digested DNA was ligated with double-stranded TeSLA adapters, and 30 pg of the ligated DNA was subsequently used for long-range PCR amplifications. Amplification reactions were carried out in 25 μl volume, using 2.5 units of FailSafe Enzyme Mix (Lucigen) with FailSafe buffer H and 0.25 μM primers (AP and TeSLA-TP). After the initial melt at 94°C for 2 min, 25 PCR cycles were carried out (94°C for 15 s, 60°C for 30 s, and 72°C for 15 min). Amplified PCR products were resolved on a 1.2% agarose gel at 50V overnight in 1X TBE buffer. Southern blotting and telomere signal detection was performed using the TeloTAGGG Telomere Length Assay kit (Roche), as described for the TRF assay. The TeSLA-QUANT software was used for image quantification and statistical analysis, as described in Lai et al. (2017) (link).

Ivanyi-Nagy R., Ahmed S.M., Peter S., Ramani P.D., Ong P.F., Dreesen O, & Dröge P. (2018). The RNA interactome of human telomerase RNA reveals a coding-independent role for a histone mRNA in telomere homeostasis. eLife, 7, e40037.

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TeSLA Telomere Length Measurement Protocol

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Genomic DNA was extracted using the Gentra Puregene DNA Extraction Kit (Qiagen) according to the manufacturer's instructions and quantified on a NanoDrop (Thermo Scientific). TeSLA measurements were performed as previously described (Lai et al, 2017 ). In brief, T4 DNA ligase (New England Biolabs), 1 mM ATP, 10 − 3 μM of TeSLA‐Ts, and 50 ng of isolated genomic DNA were mixed in 1× CutSmart buffer (New England Biolabs) and incubated at 35°C for 12–16 h. The mixture was then digested with CviAII, BfaI, NdeI, and MseI (New England Biolabs) to generate DNA fragments with 5′ AT and TA overhangs. Shrimp alkaline phosphatase (rSAP; New England Biolabs) was added to the digested mixture to remove 5′ phosphate from each DNA fragment. The mixture was combined with T4 DNA ligase, 1 mM ATP, 1 μM of AT adapter, and 1 μM of TA adapter in 1× CutSmart buffer to incubate at 16°C for 12–16 h. Multiple PCRs were then performed using FailSafe Enzyme Mix (Lucigen) with 1× FailSafe buffer H containing 0.25 μM AP/TeSLA‐TP primers and 40 pg of ligated DNA. PCR products were resolved on a 0.85% agarose gel (1.5 V/cm for 19 h). After gel electrophoresis, Southern blot is applied to detect amplified telomeres.

Sepe S., Rossiello F., Cancila V., Iannelli F., Matti V., Cicio G., Cabrini M., Marinelli E., Alabi B.R., di Lillo A., Di Napoli A., Shay J.W., Tripodo C, & d’Adda di Fagagna F. (2021). DNA damage response at telomeres boosts the transcription of SARS‐CoV‐2 receptor ACE2 during aging. EMBO Reports, 23(2), e53658.

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Inverse PCR for Ae. aegypti Transposon

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Inverse PCR was done essentially according to a published protocol [88 (link)] with modifications. Genomic DNA was digested overnight with Sau3AI and CviAII (New England Biolabs, Ipswich, MA, USA). The digested DNA was purified (Qiagen PCR purification kit) and ligated with T4 DNA ligase (New England Biolabs, Ipswich, MA, USA) and used as a template for the following PCR reaction: 98°C for 3 min., 37 cycles of 98°C for 30 s, 58°C for 45 s, 72°C for 3 min, and 1 cycle of 72°C for 5 min, using the primers listed in S1 Table. Amplification products were gel extracted with 1% agarose gel and sequenced. VectorBase (http://www.vectorbase.org) was searched for sequences corresponding to the junctions between transposon landing sites on Ae. aegypti genome and transposon Mos1 left arms using the BLASTn tool.

Dong Y., Dong S., Dizaji N.B., Rutkowski N., Pohlenz T., Myles K, & Dimopoulos G. (2022). The Aedes aegypti siRNA pathway mediates broad-spectrum defense against human pathogenic viruses and modulates antibacterial and antifungal defenses. PLoS Biology, 20(6), e3001668.

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Telomere Length Measurement by TeSLA

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TeSLA measurements were performed as described in Lai et al. (2017)^{84 (link)}. Briefly, 50 ng of genomic DNA were mixed with 2 mM ATP, 0.5 μl T4 ligase (NEB), and Telo1-6 ligation oligos at 10 nM each in CutSmart buffer 1X (NEB), and incubated overnight at 35 °C. The mixture was digested with CviAII, MseI, NdeI and BfaI (NEB) to generate DNA fragments with 5′ AT and TA overhangs. The 5’ phosphate of these DNA fragments was removed using the shrimp alkaline phosphatase (rSAP, NEB). The DNA mixture was then incubated overnight at 16 °C with T4 DNA ligase, 1 mM ATP, 1 μM of AT adapter, and 1 μM of TA adapter in 1× CutSmart buffer.
Multiple PCRs were performed using FailSafe Enzyme Mix (Lucigen) with 1× FailSafe buffer H containing 0.25 μM AP/TeSLA-TP primers and 40 pg of ligated DNA. PCR products were loaded on a 0.85% agarose gel and run for 19 h at 1.5 V/cm. After gel electrophoresis, the amplified telomeres were detected by Southern blot.

Rosso I., Jones-Weinert C., Rossiello F., Cabrini M., Brambillasca S., Munoz-Sagredo L., Lavagnino Z., Martini E., Tedone E., Garre’ M., Aguado J., Parazzoli D., Mione M., Shay J.W., Mercurio C, & d’Adda di Fagagna F. (2023). Alternative lengthening of telomeres (ALT) cells viability is dependent on C-rich telomeric RNAs. Nature Communications, 14, 7086.

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Viral Genome Sequencing Protocol

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Specific primers were designed on the viral sequences identified with VIDISCA-454 and PCRs using DreamTaq DNA polymerase (Thermo Scientific) were performed to connect fragments. Sequencing reactions were carried out with nested primers directly on the amplified products using the Big Dye terminator chemistry (BigDye^® Terminator v1.1 Cycle Sequencing Kit, Applied Biosystems).
The ends of the genome were determined using genomic fragments obtained by specific digestion with 2 different restriction enzymes (MseI and CviAII from New England Biolabs) to obtain overlapping fragments, to which VIDISCA adaptors were subsequently ligated; semi specific PCRs were then performed with a combination of one primer annealing to the known viral sequence and one to the adaptor. After sequencing the obtained amplicons, the novel sequence was used as a template for new primer design and the whole procedure was repeated until reaching the end of the genome. Specific PCRs were used as confirmation. All primers used for PCR and sequencing reactions are available upon request.

Canuti M., Williams C.V., Gadi S.R., Jebbink M.F., Oude Munnink B.B., Jazaeri Farsani S.M., Cullen J.M, & van der Hoek L. (2014). Persistent viremia by a novel parvovirus in a slow loris (Nycticebus coucang) with diffuse histiocytic sarcoma. Frontiers in Microbiology, 5, 655.

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Genomic DNA Isolation and Library Preparation for B. thuringiensis HER1410

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The genomic DNA (gDNA) of B. thuringiensis HER1410 was isolated using the DNeasy Blood and Tissue kit (Qiagen) and concentrated via ethanol precipitation [82 (link)]. The extracted gDNA was partially digested with the restriction enzyme CviAII (New England Biolabs) and subsequently 5′-dephosphorylated with Calf Intestinal Alkaline Phosphatase (New England Biolabs). CviAII cuts on a sequence motif “CATG,” which is highly frequent on bacterial genomes, with 17,370 sites in HER1410 genome, cutting on average every 356.8 base pairs (bp). The digested DNA was separated by agarose electrophoresis and an agarose block with DNA fragments spanning between about 450–750 base pairs (marker bands) was cut out, subjected to gel-extraction DNA purification with QIAquick Gel Extraction Kit (Qiagen), and purified using ethanol precipitation [82 (link)]. The DNA fragments size was chosen to meet Illumina technical requirements and according to the gene size distribution. In HER1410, 75% of the protein-coding genes are shorter than 1 kb, longer fragments might have led to an ample generation of chimeric genes. Gap filling of the 3′ overhangs and A-tailing were performed using Taq DNA polymerase. The library was subsequently cloned into the plasmid pCR^TM8/GW/TOPO^® using the pCR™8/GW/TOPO™ TA Cloning Kit (ThermoFisher).

Lechuga A., Lood C., Berjón-Otero M., del Prado A., Wagemans J., van Noort V., Lavigne R., Salas M, & Redrejo-Rodríguez M. (2021). Unraveling Protein Interactions between the Temperate Virus Bam35 and Its Bacillus Host Using an Integrative Yeast Two Hybrid–High Throughput Sequencing Approach. International Journal of Molecular Sciences, 22(20), 11105.

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