DNA was extracted using a Microbial DNA extraction kit (QIAGEN)70 (link) and 50 µl from a capsule device or 100 mg stool. The 16S rRNA amplicons were generated using Earth Microbiome Project-recommended 515F/806R primer pairs and 5PRIME HotMasterMix (Quantabio, 2200410) with the following program in a thermocycler: 94 °C for 3 min, 35 cycles of 94 °C for 45 s, 50 °C for 60 s and 72 °C for 90 s, followed by 72 °C for 10 min. PCR products were cleaned, quantified and pooled using the UltraClean 96 PCR Cleanup kit (QIAGEN, 12596-4) and Quant-iT dsDNA High Sensitivity Assay kit (Invitrogen, Q33120). Samples were sequenced with 300-bp reads on a MiSeq (Illumina). Sequence data were de-multiplexed using the Illumina bcl2fastq algorithm at the Chan Zuckerberg BioHub Sequencing facility. Subsequent processing was performed using the R statistical computing environment v.4.0.3 (ref. 71 ) and DADA2 as previously described using pseudo-pooling72 (link). truncLenF and truncLenR parameters were set to 250 and 180, respectively. Taxonomy was assigned using the Silva rRNA database v.132 (ref. 73 (link)). Samples with >2,500 reads were retained for analyses.
Ultraclean 96 pcr cleanup kit
Manufactured by Qiagen
Sourced in Germany
The UltraClean 96 PCR Cleanup Kit is a laboratory equipment product designed for the purification of PCR amplicons. It efficiently removes primers, nucleotides, enzymes, and other contaminants from PCR reactions, preparing the amplicons for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Quant it dsdna high sensitivity assay kit, by Thermo Fisher Scientific (3 mentions)
5prime hotmastermix, by Quantabio (3 mentions)
Dneasy powersoil htp 96 kit, by Qiagen (2 mentions)
Bcl2fastq algorithm, by Illumina (1 mentions)
Microbial dna extraction kit, by Qiagen (1 mentions)
Nebexpress cell free e coli protein synthesis system, by New England Biolabs (1 mentions)
Hybond n hybridisation membrane, by GE Healthcare (1 mentions)
Dneasy ultraclean 96 microbial kit, by Qiagen (1 mentions)
Dntps, by Merck Group (1 mentions)
Q5 high fidelity dna polymerase, by Merck Group (1 mentions)
7 protocols using ultraclean 96 pcr cleanup kit
1
Gut Microbiome Profiling via 16S rRNA Sequencing
2
In vitro Expression of Ehrlichia Proteins
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In vitro expression of Ehrlichia proteins was performed using the NEBExpress cell-free E. coli protein synthesis system (New England Biolabs, Ipswich, MA). Lyophilized plasmids were reconstituted in water and purified using the UltraClean 96 PCR cleanup kit (Qiagen, Germantown, MD). Plasmids were then added to E. coli extract and a reaction premix in a 96-well plate and incubated at 37°C for 3 h with orbital shaking (300 rpm) according to the manufacturer’s instructions.
3
Detection of Tobamovirus Infection from Petioles
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Five fresh-cut petioles were impressed in a one cm2 of Hybond®-N+ hybridisation membrane (GE Healthcare, Chicago, IL, USA), dried at room temperature for 5 min and placed in a 1.5 ml tube containing 0.5 ml of glycine buffer. Tubes were vortexed for 30 s and heated at 95 °C for 10 min. Three µl were used for the subsequent steps.
Finally, the 42 sample preparations were tested by DAS-ELISA using a commercial kit of polyclonal antibodies for TMV, which can also detect ToBRFV (AGDIA, Elkhart, IN, USA). The same samples were also tested by end-point RT-PCR using the primers ToBRFV-F-5722 and ToBRFV-R-6179, and by a real-time RT-PCR-MGB-probe based-method according to the protocols described previously.
In order to understand if the samples with high Ct value were really positive or false positives, the products obtained with the samples named 1A, 3C, 7R, 8R and 9R using the methods No. 3 and No. 4 of sample preparation were analysed in 2% agarose gel. The products were purified with the Ultraclean 96 PCR Cleanup Kit (Qiagen, Hilden, Germany) according to the manufacture’s instruction and sequenced in both directions using an ABI PRISM 3100 DNA sequence analyser (Applied Biosystems, Foster City, CA, USA).
Finally, the 42 sample preparations were tested by DAS-ELISA using a commercial kit of polyclonal antibodies for TMV, which can also detect ToBRFV (AGDIA, Elkhart, IN, USA). The same samples were also tested by end-point RT-PCR using the primers ToBRFV-F-5722 and ToBRFV-R-6179, and by a real-time RT-PCR-MGB-probe based-method according to the protocols described previously.
In order to understand if the samples with high Ct value were really positive or false positives, the products obtained with the samples named 1A, 3C, 7R, 8R and 9R using the methods No. 3 and No. 4 of sample preparation were analysed in 2% agarose gel. The products were purified with the Ultraclean 96 PCR Cleanup Kit (Qiagen, Hilden, Germany) according to the manufacture’s instruction and sequenced in both directions using an ABI PRISM 3100 DNA sequence analyser (Applied Biosystems, Foster City, CA, USA).
4
Fecal Microbiome 16S rRNA Sequencing
5
16S rRNA Amplicon Sequencing from Fecal/Culture
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DNA from fecal samples or 50 μL of saturated bacterial cultures were extracted using an extraction kit such as the DNeasy UltraClean 96 Microbial Kit (Qiagen, 10196-4). Three microliters of extracted gDNA were used for PCR in 75-μL volumes containing Earth Microbiome Project-recommended 515F/806R primer pairs (0.4 μM final concentration) and a polymerase such as that of the 5PRIME HotMasterMix (Quantabio, 2200410) to generate V4 region 16S rRNA amplicons. The following thermocycler conditions were used: 94°C for 3 min, 35 cycles of [94°C for 45 s, 50°C for 60 s, and 72°C for 90 s], then 72°C for 10 min. PCR products were individually cleaned up and quantified using the UltraClean 96 PCR Cleanup Kit (Qiagen, 12596-4) and the Quant-iT dsDNA High Sensitivity Assay kit (Invitrogen, Q33120) before 200 ng of PCR product for each sample were manually pooled. Pooled libraries were then sequenced with 250- or 300-bp paired-end reads on a MiSeq (Illumina).
6
16S rRNA Gene Amplification and Sequencing Protocol
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PCR amplification of the 16S rRNA gene V4 region was performed according to method of Caporaso et al. [68 (link)]. Samples were amplified in triplicate using 0.25 μl Q5 High-Fidelity DNA Polymerase (NEB) per 25 μl reaction with 0.2 mM dNTPs (Sigma), 1X Q5 Reaction Buffer, 200 pM 515F primer (IDT), and 200 pM barcoded 806R primer (IDT) in PCR-Clean water (MoBio). A water-template negative control reaction was included with each sample to confirm absence of contaminating DNA. Samples were amplified at 98°C for 30 seconds, followed by 35 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for 20 s with a final 2 min extension at 72°C. Triplicate reactions were pooled and then separated on an agarose gel in parallel with the paired water control reactions to confirm successful amplification. Individual samples were pooled and purified using an UltraClean 96 PCR Cleanup Kit (Qiagen). The pooled library was then quantified, diluted, supplemented with 10% PhiX, chemically and heat denatured as per Illumina MiSeq protocols, and sequenced at 10 pM on an Illumina V2 1x300 sequencing kit using custom sequencing primers as per Caporaso [68 (link)].
7
Profiling Gut Bacterial Diversity
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Total DNA was extracted from frozen fecal pellets using the PowerSoil-htp 96 well DNA isolation Kit (MoBio) or the DNeasy PowerSoil HTP 96 Kit (QIAGEN). Barcoded primers were used to amplify the V3-V4 region of the 16S rRNA gene from extracted bacterial DNA using primers 515fB and 806rB via PCR (EMP). PCR clean-up was performed with UltraClean PCR Clean-Up Kit (MoBio) or UltraClean 96 PCR Cleanup Kit (QIAGEN) before quantification of amplicon products with Quant-iT dsDNA Assay Kit, high sensitivity (Invitrogen). Amplicons were pooled by adding 100 ng of each product. Illumina MiSeq paired-end reads runs were performed, with experiments divided among runs such that no more than 384 samples were read per run.
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