Metagenomic libraries (mNGS) were prepared and quantified essentially as described [13 (link)]. Briefly, total nucleic acid was concentrated to 10 μl with RNA Clean and Concentrator-5 spin columns (Zymo Research, CA) and RNA was reverse transcribed with random primers using Superscript III (SSRTIII) 1st Strand reagents (Life Technologies), followed by 2nd strand synthesis with Sequenase V2.0 T7 DNA pol (Affymetrix). Double stranded DNA/cDNA was recovered with DNA Clean and Concentrator-5 spin columns (Zymo Research) and -barcoded with Nextera XT indices lacking 5’ biotin tags using 24 cycles of amplification (IDT, Coralville IA; Illumina, Carlsbad CA). Nextera libraries were purified with Agencourt AMPpure XP beads (Beckman Coulter) and quantified by a 2200 TapeStation (Agilent) and Qubit fluorometer (Life Technologies).
Dna clean and concentrator 5 spin columns
Manufactured by Zymo Research
Sourced in United States
The DNA Clean and Concentrator-5 spin columns are a laboratory product designed to purify and concentrate DNA samples. The core function of these spin columns is to efficiently remove unwanted substances, such as salts, primers, and enzymes, from DNA samples, while simultaneously concentrating the DNA for downstream applications.
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Lab products found in correlation
Superscript 3 ssrtiii 1st strand reagents, by Thermo Fisher Scientific (1 mentions)
Sequenase v2.0 t7 dna pol, by Thermo Fisher Scientific (1 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Rna clean and concentrator 5 spin column, by Zymo Research (1 mentions)
3730 dna analyzer, by Thermo Fisher Scientific (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
2 protocols using dna clean and concentrator 5 spin columns
1
Metagenomic Library Preparation and Quantification
2
Unnatural Base Pair Amplification
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All PCR amplifications were performed in a CFX Connect Real-Time PCR Detection System (Bio-Rad), in a total volume of 25 μl using the following conditions: 1× OneTaq reaction buffer, 0.5× Sybr Green I, MgSO4 adjusted to 4.0 mM, 0.2 mM of each dNTP, 50 μM of each unnatural triphosphate, 1 μM of Primer1 and Primer2 (See Supplementary Table S2) and 0.02 U/μl of the DNA polymerase. Other conditions specific for each round of screening are described in Supplementary Table S3. Amplified products were purified using DNA Clean and Concentrator-5 spin columns from Zymo Research (Irvine, CA, USA). After purification, the PCR products were sequenced on a 3730 DNA Analyzer (Applied Biosystems) to determine the retention of the UBP as described in the Supplementary Material. Fidelity was characterized from UBP retention as determined by sequencing with Primer1 on a 3730 DNA Analyzer (Applied Biosystems).
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