The remaining RNA was used to obtain cDNA and amplified through a Single-primer isothermal amplification (SPIA) approach using the Ovation RNA-seq v2 kit (NuGEN, San Carlos, CA, USA) according to the manufacturer’s instructions. The SPIA products were purified using Genjet PCR purification kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions before being sequenced at SciLifeLab/Genome Center (Uppsala, Sweden) using the Ion S5 XL system (Thermo Fisher, Waltham, MA, USA) and a 530 chip.
Genjet pcr purification kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Genjet PCR purification kit is a product designed to purify PCR amplicons. It utilizes a simple and efficient silica-membrane-based technology to remove unwanted reagents, primer dimers, and non-specific amplification products from PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Ovation rna seq v2 kit, by Tecan (1 mentions)
Ion s5 xl system, by Thermo Fisher Scientific (1 mentions)
Longamp taq reaction buffer, by New England Biolabs (1 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (1 mentions)
Nextera xt library preparation kit, by Illumina (1 mentions)
Miseq reagent kit, by Illumina (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Genejet genomic dna purification kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using genjet pcr purification kit
1
Single-primer isothermal RNA-seq amplification
2
Amplification and Sequencing of 2480 bp DNA Fragment
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A 2480 bp fragment was amplified using LongAmp Taq DNA Polymeranse (2.5 U), dNTP mix (0.33 mM), 1× LongAmp Taq Reaction buffer (New England BioLabs, M0323S, Ipswich, MA, USA), 0.4 μM of respective primer Sp1F and Sp10R (Table A2), and 3 μl of template. Thermocycling conditions were 94 °C for 30 s and then 40 cycles of 94 °C for 30 s, 64 °C for 45 s, elongation time at 65 °C for 220 s, and finally 65 °C for 10 min. The PCR products were purified using the GenJET PCR Purification kit (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing libraries were made using Nextera XT Library Preparation Kit (Illumina, San Diego, CA, USA), normalization and pooling of 2 nM sequencing libraries was done based on concentration measurements from Agilent High Sensitivity DNA Kit (2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA, USA). The pool of sequencing libraries was denaturated with NaOH and further diluted with hybridization buffer to a final concentration of 10 pM and spiked with 5% PhiX for diversity. Paired-end sequencing was performed with MiSeq Reagent Kit v3 600 cycles on the MiSeq instrument (Illumina, San Diego, CA, USA) at the National Veterinary Institute, Uppsala, Sweden.
3
Identification of Preserved Bacterial Strains
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With the aim to tentatively identify the preserved strains, the isolates were revitalized from −80 °C on RAE medium with 1% of ethanol and 1% of acetic acid. A single colony from each culture was inoculated onto the same medium and grown for three days to obtain enough biomass for DNA isolation. After biomass harvesting, the DNA was isolated by the GeneJET Genomic DNA Purification Kit (Thermo Scientific, Waltham, MA, USA). The 16S–23S rRNA gene ITS regions were amplified by the primers SpaFw (5′-TGCGG(T/C)TGGATCACCTCCT-3′) and SpaRev (5′-GTGCC(A/T)AGGCATCCACCG- 3′), and the 16S rRNA gene sequence by the primers 27f (5′-AGAGTTTGATCMTGGCTCAG-3′) and rH1542 (5′-AAGGAGGTGATCCAGCCGCA-3′). The PCR-products were purified with the GenJet PCR Purification Kit (Thermo Scientific, Waltham, Massachusetts, USA) and sequenced by the Sanger method at Microsynth (Vienna, Austria).
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