All oligonucleotides used in this study as crRNA or DNA primers are listed in Table S1 and were ordered from biomers.net GmbH (Ulm, Germany). The DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) was used for extraction of genomic DNA of apple tissue and the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) for isolation of plasmid DNA from bacterial clones. The Invitrogen Qubit dsDNA BR Assay Kit was used for quantification of DNA with the Invitrogen Qubit 4 Fluorometer (both supplied by Thermo Fisher Scientific Inc., Waltham, MA, USA). PCR amplifications were performed using the Phusion High-Fidelity PCR Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) or the Type-it Microsatellite PCR Kit (Qiagen GmbH, Hilden, Germany). PCR products were purified with the Roche High Pure PCR Product Purification Kit (Merck KGaA, Darmstadt, Germany). Clonings were performed using the TOPO XL-2 Complete PCR Cloning Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The kits were used according to the manufacturer’s recommendations, and the specifications are given below.
Topo xl 2 complete pcr cloning kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TOPO XL-2 Complete PCR Cloning Kit is a laboratory product designed for the cloning of PCR amplified DNA fragments. The kit includes all the necessary components for the efficient ligation and transformation of PCR products into bacterial hosts.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy plant mini kit, by Qiagen (2 mentions)
Qiaprep spin miniprep kit, by Qiagen (2 mentions)
Phusion high fidelity pcr kit, by Thermo Fisher Scientific (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Invitrogen qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Type it microsatellite pcr kit, by Qiagen (1 mentions)
Fish tag dna multicolor kit, by Thermo Fisher Scientific (1 mentions)
Platinum taq dna polymerase kit, by Thermo Fisher Scientific (1 mentions)
Superscript 3 one step rt pcr system with, by Thermo Fisher Scientific (1 mentions)
Monarch dna gel extraction kit, by New England Biolabs (1 mentions)
Primestar max dna polymerase, by Takara Bio (1 mentions)
Amv reverse transcriptase, by Promega (1 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Precellys homogenizer, by Bertin Technologies (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
6 protocols using topo xl 2 complete pcr cloning kit
1
DNA Extraction and Quantification Protocol
2
Fluorescent FISH Probe Generation
3
One-Step RT-PCR Virus Sequencing Protocol
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For further analysis, all positive samples were subjected to one-step RT-PCR (Reverse Transcription Polymerase Chain Reaction) using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase Kit, Invitrogen, Carlsbad, CA, USA. In this procedure, we combined both cDNA synthesis (complementary DNA) and PCR amplification in a single tube and used genome-specific primers for all virus fragments: MBTuni-12 [5-ACGCGTGATCAGCAAAAGCAGG] and MBTuni-13 [5-ACGCGTGATCAGTAGTAGAAACAAGG]. Subsequently, the isolated fragments were cloned into the TOPO vector using the TOPO XL-2 Complete PCR Cloning Kit from Invitrogen and then sequenced with M13 primers using the Sanger method. The Sanger reads were assembled with the SnapGene v.2.3.2 software. For segments longer than the Sanger reads, sequencing was performed in several steps. After sequencing with M13 primers, the primers were synthesized for the further sequencing of the remaining part of the segment.
4
Validating Tetmemena MIC Genome Assembly
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The complex Russian doll locus on MIC contig TMEMEN_MIC_21461 in Tetmemena was validated by PCR to confirm the Tetmemena MIC genome assembly. Tetmemena micronuclear DNA was purified as described previously and used as template for PCR using PrimeSTAR Max DNA polymerase (Takara Bio). 11 primer sets (Supplementary file 14 ) were designed to amplify products between 3 kb and 6 kb in length, with overlapping regions between consecutive primer pairs. The resulting PCR products were visualized through agarose gel electrophoresis, and bands of the expected size were extracted using a Monarch DNA Gel Extraction Kit (New England Biolabs). The purified gel bands were cloned using a TOPO XL-2 Complete PCR Cloning Kit (Invitrogen), transformed into One Shot OmniMAX 2 T1R E. coli cells (Invitrogen), and individual clones were grown and their plasmids harvested with a QIAprep Spin Miniprep Kit (QIAGEN). The plasmid ends were Sanger sequenced, as well as the region where the Oxytricha MIC assembly contains inserted MDSs (Genewiz). Sanger sequencing reads were mapped to the Tetmemena MIC contig TMEMEN_MIC_21461 and visualized using Geneious Prime 2021.1.1 (https://www.geneious.com ).
5
Molecular Characterization of PEDV Strains
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Viral RNA was extracted from intestinal contents using an RNeasy Mini Kit (QIAGEN, USA). Reverse transcription and amplification of the PEDV S gene were carried out using AMV reverse transcriptase (Promega, USA) and PrimeSTAR® GXL DNA polymerase (TaKaRa, Dalian, China), respectively, with the primers 5´-GCAACACTATGCATGCCAAT-3´ and 5´-TGTTGCACACTTATTGGCAGG-3´. PCR products were isolated by agarose gel electrophoresis and then purified using a DNA Gel Extraction Kit. The target fragment was cloned into the vector included in the TOPO™ XL-2 Complete PCR Cloning Kit (Invitrogen, USA) and the identity of the plasmids was confirmed by restriction enzyme digestion and sequencing. The sequence was assembled and analyzed using DNAStar 7.0 and BioEdit software, respectively. Forty-eight PEDV reference strains that contained five different PEDV genotypes (Ia, Ib, IIa, IIb and S-INDEL) were obtained from the GenBank database for phylogenetic analysis. A phylogenetic tree was constructed with MEGA6.0 software using the neighbor-joining method with bootstrap values calculated for each node from 1,000 replicates.
6
Genomic DNA Extraction and Amplification
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All samples were homogenized with a Precellys® homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). Genomic DNA was obtained using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and quality was assessed visually by agarose gel electrophoresis. In order to obtain longer fragments upon amplification of the md-MT genes, the PlatinumTM SupferFiTM Green PCR Master Mix (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was applied for PCR amplification (for primers and PCR setup, see Table S2 ). PCR products were separated on a 1.5% agarose gel (Biozym, Hessisch Oldendorf, Germany) by electrophoresis. Gene-specific bands were excised, purified with the QIAquick™ Gel Extraction Kit (Qiagen, Hilden, Germany) and cloned with the TOPO™ XL-2 Complete PCR Cloning Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) for Sequencing (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Insert-containing plasmids were purified using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) and sent to Microysnth AG (Balgach, Switzerland) for Sanger sequencing.
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