Microbial community profiles were assessed by sequencing the 16S rRNA gene. The first step was the amplification of V3 and V4 variable fragments of analyzed gene according to the protocol recommended by Illumina (San Diego, CA, USA). Primers with overhanging adapters compatible with Illumina indexes and sequencing adapters in paired-end sequencing technique were used. Kappa HiFi polymerase (Roche, Mannheim, Germany) was used to amplify fragment with an average 464 bp length. Next the specificity of obtained products was evaluated in an agarose gel and then purified on AMPure XP magnetic beads (Beckman Coulter, CA, USA). The indexing reactions were also carried out using the Kappa HiFi polymerase (Roche, Mannheim, Germany) with the Nextera XT dual-index set (Illumina, San Diego, CA, USA). The concentration of the obtained libraries was determined using the Qubit 2.0 device (Thermo Fisher Scientific, Waltham, MA, USA) and pooled in equal concentrations. The library thus prepared was sequenced on the Miseq platform (Illumina, San Diego, CA, USA) using the kit (MiSeq Reagent Kit v3, 600 cycles).
Kappa hifi polymerase
Manufactured by Roche
Sourced in Germany
The Kappa HiFi polymerase is a high-fidelity DNA polymerase designed for accurate DNA amplification. It has a proofreading activity that reduces the error rate during DNA synthesis, making it suitable for applications requiring precise replication of DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 550 system, by Illumina (1 mentions)
Sureselectqxt target enrichment protocol, by Agilent Technologies (1 mentions)
Nextera xt dna library preparation kit, by Illumina (1 mentions)
Miseq system, by Illumina (1 mentions)
Maxwell rsc instrument, by Promega (1 mentions)
Qubit 2.0 device, by Thermo Fisher Scientific (1 mentions)
Nextera xt dual index set, by Illumina (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Miseq platform, by Illumina (1 mentions)
Ampure xp magnetic beads, by Beckman Coulter (1 mentions)
Genemorph 2 random mutagenesis kit, by Agilent Technologies (1 mentions)
Erythromycin, by Merck Group (1 mentions)
Phusion polymerase, by New England Biolabs (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Chem-Impex (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Expand high fidelity polymerase, by Roche (1 mentions)
Pfx50 dna polymerase, by Thermo Fisher Scientific (1 mentions)
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (1 mentions)
6 protocols using kappa hifi polymerase
1
Profiling Microbial Communities via 16S rRNA Sequencing
2
Mitochondrial Genome Sequencing Protocol
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DNA isolation was performed from peripheral blood with a semi-automatic Maxwell RSC Instrument (Promega, Madison, WI, USA). The library was prepared via the Agilent SureSelectQXT Target Enrichment protocol using a custom gene panel with mtDNA enrichment in accordance with the manufacturer’s instructions. Paired-end sequencing was performed in a NextSeq550 System (Illumina, San Diego, CA, USA) at 2 × 150 bp.
The potential contribution of frequent NUMTs insertions for the heteroplasmic variants selected for further analysis was determined via additional sequencing based on long-range PCR. The mitochondrial genome was amplified in one long amplicon (left primer 5′-GGACACTAGGAAAAAACCTTGTAGAGAGAGAGAGAGAG-3′; right primer 5′-AAAGAGAGCTGTTCCTTTGGACTAACA-3′) using Kappa HiFi polymerase (Roche, Basel, Switzerland). The products were then evaluated on an agarose gel. The library was prepared using a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) according to the standard protocol. Paired-end sequencing was performed in a Miseq System (Illumina, San Diego, CA, USA) at 2 × 250 bp.
The potential contribution of frequent NUMTs insertions for the heteroplasmic variants selected for further analysis was determined via additional sequencing based on long-range PCR. The mitochondrial genome was amplified in one long amplicon (left primer 5′-GGACACTAGGAAAAAACCTTGTAGAGAGAGAGAGAGAG-3′; right primer 5′-AAAGAGAGCTGTTCCTTTGGACTAACA-3′) using Kappa HiFi polymerase (Roche, Basel, Switzerland). The products were then evaluated on an agarose gel. The library was prepared using a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) according to the standard protocol. Paired-end sequencing was performed in a Miseq System (Illumina, San Diego, CA, USA) at 2 × 250 bp.
3
Molecular Cloning and Mutagenesis Protocol
4
Thermophilic Lipase LipJ Cloning
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From the consensus sequence obtained, new degenerate primers LipBFwInNcoI/LipBFwOutHindIII (Table 2 ) were designed and used for amplification (Expand High Fidelity polymerase, Roche) of a putative thermophilic lipase, designated LipJ, using JR3 genomic DNA as a template. The resulting PCR fragment was ligated to pGEM-T® Easy vector (Promega) and transformed into E. coli DH5α. For overexpression, pGEMT-LipJ was digested with NcoI/HindIII, ligated to the doubly digested expression vector pET28a and transformed in E. coli BL21 star (DE3). Additionally, lipJ gene was amplified (Kappa-HiFi polymerase, Kapa Biosystems) using specific primers FwLipJTOPO/BwLipJTOPO, and ligated to pET101/D-TOPO, producing recombinant plasmid pET101-LipJ-Histag, which contains the full-length enzyme linked to a C-terminal His6-tag. All constructions were verified by sequencing. The DNA sequence of lipJ was submitted to NCBI and given the GenBank Accession Number KU747177.
5
Screening Wolbachia Infection in Drosophila
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DNA for polymerase chain reaction (PCR) screening of Wolbachia infection status was prepared from single flies by placing individual males in a standard fly squish buffer (50 μl of 1M Tris pH 8.0, 0.5M EDTA, 5M NaCl) plus 1 μl of 10 mg/ml Proteinase K. Flies were then placed in a thermocycler at 37° for 30 min, 95° for 2 min followed by a 4° hold. PCR was performed using 4 μl of fly squish product in a total volume of 50 μl. The presence of Wolbachia was confirmed by PCR using two sets of primers: (i) Wolbachia_F2 (5′-TGGCTCACATAGATGCTGGT- 3′) and Wolbachia_R2 (5′-GTCCCATTTCTCACGCATTT-3′); and (ii) Wolbachia_F3 (5′-ATCCTGCAAATTGGCGTACT-3′) and Wolbachia_R3 (5′-ATAACGCACACCTGGCAAAT-3′). To ensure DNA preparation was sufficient for PCR amplification, control primers were used from the D. melanogaster genome: rDNA-F (5′-AAACTAGGATTAGATACCCTATTAT-3′) and rDNA-R (5′-AAGAGCGACGGGCGATGTGT-3′). PCR was performed with Kappa HiFi polymerase (KAPA Biosystems, KK2502) using the following reaction conditions: 30 cycles of 95° for 20 sec, 60° for 15 sec, and 72° for 90 sec.
6
Murine Mbnl1 e1 Minigene Construction
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Min_e1_WT minigene was prepared by subcloning a ∼2.0 kb murine genomic Mbnl1 fragment spanning e1 with parts of flanking introns into human cTNT minigene [(46 (link)), a kind gift from M. Disney; The Scripps Research Institute]. Mbnl1 fragment was amplified from C2C12 genomic DNA using a two-step nested PCR with F16/R16 and F17/R17 primers. The second round PCR product was cloned into cTNT minigene backbone with BamHI and SalI, replacing alternative cTNT e5. Min_e1_Mut3 was prepared using a three-step PCR reaction. First, two PCR reactions were performed to introduce point mutations abolishing MBNL1 binding in e1 with F17/R18 and F18/R17 primers. Both PCR products were annealed and extended and then subjected to PCR 3 with F17/R17 primers. The ∼2.0 kb PCR product was cloned into BamHI/SalI digested cTNT vector.
All clones and PCR bands were verified by sequencing. PCR fragments used for cloning were amplified with either Platinum Taq DNA Polymerase High Fidelity, Pfx50 DNA polymerase (Invitrogen), or Kappa HiFi polymerase (Kapa Biosystems). F/R sequences are listed inSupplementary Table S1 .
All clones and PCR bands were verified by sequencing. PCR fragments used for cloning were amplified with either Platinum Taq DNA Polymerase High Fidelity, Pfx50 DNA polymerase (Invitrogen), or Kappa HiFi polymerase (Kapa Biosystems). F/R sequences are listed in
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