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Sau3ai

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Sau3AI is a Type II restriction endonuclease enzyme that cleaves DNA at the recognition sequence 5'-GATC-3'. It is commonly used in molecular biology applications such as DNA digestion and fragment analysis.

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Lab products found in correlation

Bamhi, by New England Biolabs (4 mentions) T4 dna ligase, by New England Biolabs (4 mentions) T4 dna ligase, by Promega (2 mentions) Proteinase k, by Thermo Fisher Scientific (2 mentions) Qiaex 2, by Qiagen (1 mentions) Calf intestinal alkaline phosphatase, by New England Biolabs (1 mentions) Bacteria gen dna kit, by CWBIO (1 mentions) Mluci, by New England Biolabs (1 mentions) Bglii, by New England Biolabs (1 mentions) Dneasy plant mini kit, by Qiagen (1 mentions) Isoamyl alcohol, by Merck Group (1 mentions) Alkaline phosphatase, by New England Biolabs (1 mentions) Geraniol, by Merck Group (1 mentions) Citronellol, by Merck Group (1 mentions) Phanta super fidelity dna polymerase, by Vazyme (1 mentions) Cinnamyl alcohol, by Merck Group (1 mentions) Calf intestinal cip, by New England Biolabs (1 mentions) T4 dna ligase, by Thermo Fisher Scientific (1 mentions) P nitrophenol, by Merck Group (1 mentions) Calf intestinal phosphatase, by New England Biolabs (1 mentions)

29 protocols using sau3ai

1

Partial Digestion and Size Selection of C. pseudotuberculosis Genomic DNA

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Approximately 1 μg of genomic DNA of C. pseudotuberculosis CBO 28033 was used for each partial digestion reaction with the enzyme Sau3AI (New England Biolabs®) at 0.7 U/μl (incubated at 37 °C for 16 h in a total reaction volume of 50 μl with 1X NEBuffer 1.1). The reactions were fractionated on 0.8% agarose gel and stained with ethidium bromide. Afterwards, 1000–5000 bp DNA fragments were excised and these were purified with the QIAEX II (Qiagen®) kit.
Galvão C.E., Fragoso S.P., de Oliveira C.E., Forner O., Pereira R.R., Soares C.O, & Rosinha G.M. (2017). Identification of new Corynebacterium pseudotuberculosis antigens by immunoscreening of gene expression library. BMC Microbiology, 17, 202.
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2

Robust ezRAD Library Preparation

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ezRAD libraries22 (link) were generated following the protocol of Knapp et al. (2016). Briefly, all samples were adjusted to approximately 1 µg of DNA in 25 µl based on the AccuBlue microplate readings prior to digestion by either dilution or concentration via evaporation with a speed-vac at room temperature. Genomic DNA was digested using the isoschizomer restriction enzymes MboI and Sau3AI (New England BioLab), which both cleave at GATC recognition sites. Digestions were performed in 50 µl reactions consisting of: 18 µl HPLC grade water, 5 µl Cutsmart Buffer (provided with restriction enzyme), 1 µl MboI (10 units), 1 µl Sau3AI (10 units) and 25 µl dsDNA (~1 µg) with the following thermocycler profile: 37 °C for 3 hours, then 65 °C for 20 mins. All digested samples were then cleaned using Beckman Coulter Agencourt AMPure XP purification beads at a 1:1.8 (DNA:beads) ratio following the standard protocol. The digests were run on a 1% agarose gel (as above) and were considered fully digested when there was a smear with little to no DNA above 5,000 bp.
Johnston E.C., Forsman Z.H., Flot J.F., Schmidt-Roach S., Pinzón J.H., Knapp I.S, & Toonen R.J. (2017). A genomic glance through the fog of plasticity and diversification in Pocillopora. Scientific Reports, 7, 5991.
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3

Construction of X. oryzae pv. oryzicola Genomic Library

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A genomic library of X. oryzae pv. oryzicola strain RS105, which contains one or more effectors suppressing the resistance in rice, was constructed in the cosmid vector pHM1 [11] (link). The genomic DNA of RS105 was purified with BacteriaGen DNA kit (CWBIO, China). Then, 1 µg of DNA was partially digested with Sau3AI (NEB, USA). The vector pHM1 was digested to completion with BamHI (NEB, USA) and treated with calf intestinal alkaline phosphatase (NEB, USA). The genomic and vector DNA fragments were ligated with T4 DNA ligase (NEB, London) and transformed into DH5α. White colonies were selected on LB agar plates containing X-gal, IPTG and spectinomycin. For the library, more than 1000 clones were picked out with toothpicks and stored in three 384-well plates at −80°C. The size of the cloned genomic fragments ranged from 12 to 20 kb, and the average length was 17 kb, with approximately 3 times the coverage of the whole RS105 genome. The plasmids of each clone was purified and introduced into PXO99A one by one.
Liu H., Chang Q., Feng W., Zhang B., Wu T., Li N., Yao F., Ding X, & Chu Z. (2014). Domain Dissection of AvrRxo1 for Suppressor, Avirulence and Cytotoxicity Functions. PLoS ONE, 9(12), e113875.
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4

Hi-C Sequencing of Human Fecal Microbiome

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The Human Fecal Microbiome Dataset is a published data set sequenced using the ProxiMeta Hi-C kit (Press et al., 2017 (link)). The NCBI BioProject accessing ids are: PRJNA413092, Accession: SRR6131122, SRR6131123, and SRR6131124. This library contained two different Hi-C libraries that were constructed due to the use of two different restriction enzymes (SRR6131122, SRR6131124), MluCI and Sau3AI (New England Biolabs, Ipswich, MA, USA). Intermediate data can be found at https://doi.org/10.5281/zenodo.8226572.
Ho H., Chovatia M., Egan R., He G., Yoshinaga Y., Liachko I., O’Malley R, & Wang Z. (2023). Integrating chromatin conformation information in a self-supervised learning model improves metagenome binning. PeerJ, 11, e16129.
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5

Comparative DNA Methylation Analysis

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The DNA methylation status of SV35-T23 and SV35-RMKO was determined by digesting 800 ng of genomic DNA with BamHI, BclI, BglII, MboI, or Sau3AI from New England Biolabs for 2 h according to the manufacturer’s instructions, and digested DNAs were visualized on a 1% agarose gel.
Eutsey R.A., Powell E., Dordel J., Salter S.J., Clark T.A., Korlach J., Ehrlich G.D, & Hiller N.L. (2015). Genetic Stabilization of the Drug-Resistant PMEN1 Pneumococcus Lineage by Its Distinctive DpnIII Restriction-Modification System. mBio, 6(3), e00173-15.
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6

Characterization of CHS Gene Accessibility

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The DNA accessibility for restriction was established in the control and W.92 transgenic plants with stabilized modulation of CHS gene expression (Lorenc-Kukula et al., 2005 (link)) by using restriction enzymes AatII, PvuI, SacII, and Sau3AI (New England Biolabs). The first three enzymes were selected on the basis of the predicted cut sites in the vicinity of the +1,273 -CCGG- motif (NEB cutter V2.0). Sau3AI was selected due to its ability to digest the DNA at frequently occurring GATC sites. The genomic DNA was isolated using the DNeasy Plant Mini Kit (Qiagen) according to the manufacturer's protocol. The DNA was digested by particular enzymes for 1 h in the appropriate conditions (temperature, buffer) according to the attached protocols. The genomic DNA digested by the restriction enzymes and undigested DNA were used as templates for the real-time PCR reaction. For the analysis the +1,273 -CCGG- motif was selected on the basis of the methylation pattern experiment. The values were presented as relative quantity (RQ) in reference to the Linola control flax, set as 1.
Dzialo M., Szopa J., Czuj T, & Zuk M. (2017). Oligodeoxynucleotides Can Transiently Up- and Downregulate CHS Gene Expression in Flax by Changing DNA Methylation in a Sequence-Specific Manner. Frontiers in Plant Science, 8, 755.
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7

DNA Amplification and Enzymatic Analysis

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Phanta super-fidelity DNA polymerase (Vazyme, Nanjing, China) was used for DNA amplification. Alkaline phosphatase, calf intestinal (CIP) and restriction enzymes NotI and Sau3AI were purchased from New England BioLabs (Ipswich, MA, USA). Other restriction enzymes and T4 DNA ligase were supplied by MBI Fermentas (Baltimore, MD, USA). The p-nitrophenyl ester series, p-nitrophenol, citronellol, geraniol, cinnamyl alcohol, and isoamyl alcohol were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other commercially available chemicals and solvents were of analytical or higher grade.
Gao W., Wu K., Chen L., Fan H., Zhao Z., Gao B., Wang H, & Wei D. (2016). A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters. Microbial Cell Factories, 15, 41.
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8

Genotyping tln1 Mutant Zebrafish

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The gRNAs used to target tln1 are listed in Supplementary Table 1. The isolated mutant fish harbor a 4 bp deletion in tln1 exon 2 resulting in a frame shift that introduces a premature stop codon. tln1d4 mutant embryos recapitulate the previously described tln1 phenotype such as partially penetrant cardiac edema and embryonic lethality60 (link). Note that tln1d4 mutant fish can be kept as homozygous adults in the presence of the BAC transgene TgBAC(tln1:tln1-YPet) which rescues the lack of tln1 activity. The tln1d4 allele was genotyped by amplifying the locus through PCR and digestion of the amplicon with the restriction enzyme Sau3AI (New England Biolabs, R0169L). The digest yields a 122 bp and a 53 bp fragment for the tln1 wild-type allele and in a 171 bp fragment for the tln1d4 allele. The primers used for genotyping by PCR are listed in the Supplementary Table 1.
Note that the copy number of the tln1 wild-type and the tln1d4 alleles with TgBAC(tln1:tln1-YPet) can be determined by the intensity of the bands of the digested amplicons on a 3% agarose gel with the above genotyping protocol.
Yamaguchi N., Zhang Z., Schneider T., Wang B., Panozzo D, & Knaut H. (2022). Rear traction forces drive adherent tissue migration in vivo. Nature cell biology, 24(2), 194-204.
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9

Genotyping tln1 Mutant Zebrafish

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The gRNAs used to target tln1 are listed in Supplementary Table 1. The isolated mutant fish harbor a 4 bp deletion in tln1 exon 2 resulting in a frame shift that introduces a premature stop codon. tln1d4 mutant embryos recapitulate the previously described tln1 phenotype such as partially penetrant cardiac edema and embryonic lethality60 (link). Note that tln1d4 mutant fish can be kept as homozygous adults in the presence of the BAC transgene TgBAC(tln1:tln1-YPet) which rescues the lack of tln1 activity. The tln1d4 allele was genotyped by amplifying the locus through PCR and digestion of the amplicon with the restriction enzyme Sau3AI (New England Biolabs, R0169L). The digest yields a 122 bp and a 53 bp fragment for the tln1 wild-type allele and in a 171 bp fragment for the tln1d4 allele. The primers used for genotyping by PCR are listed in the Supplementary Table 1.
Note that the copy number of the tln1 wild-type and the tln1d4 alleles with TgBAC(tln1:tln1-YPet) can be determined by the intensity of the bands of the digested amplicons on a 3% agarose gel with the above genotyping protocol.
Yamaguchi N., Zhang Z., Schneider T., Wang B., Panozzo D, & Knaut H. (2022). Rear traction forces drive adherent tissue migration in vivo. Nature cell biology, 24(2), 194-204.
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10

Construction of genomic libraries for P. aeruginosa

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Genomic libraries were prepared as previously described (Houot et al., 2012 (link)). Briefly, P. aeruginosa PAO1 genomic DNA was purified using a DNeasy blood and cell culture kit (Qiagen, Valencia, CA) and then partially digested with 10 to 10−8 units of Sau3AI (New England Biolabs). Digested DNA was separated on a 1% agarose gel to verify the sizes of the digested products and samples containing DNA fragments from 250 to 5000 bp were purified using a QIAquick PCR purification kit (Qiagen). The pUT18 and pUT18C plasmids were digested with BamHI (New England Biolabs), dephosphorylated with calf intestinal phosphatase (New England Biolabs), and ligated to the digested PAO1 gDNA. The resulting ligation mixtures were introduced into E. coli BTH101 by heat-shock. Transformants were collected by pooling colonies, and plasmids were isolated using a QIAprep spin miniprep kit (Qiagen). The average size of the cloned library DNA was 700 bp (as determined by PCR analysis of 80 randomly-selected clones).
Orillard E, & Watts K.J. (2020). DECIPHERING THE CHE2 CHEMOSENSORY PATHWAY AND THE ROLES OF INDIVIDUAL CHE2 PROTEINS FROM PSEUDOMONAS AERUGINOSA. Molecular microbiology, 115(2), 222-237.
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