The Nextera XT Index Kit v2 (Illumina) was used for index PCR to attach the dual indices and Illumina sequencing adapters to purified 16S amplicons as previously described. Each batch of indexing reactions included a DNA/RNA-free water as a negative control. The index PCR products were cleaned using AMPure XP beads, followed by library purity analysis using the QIAxcel Advanced System (Qiagen). The index libraries were then quantified using the Qubit dsDNA HS Assay Kit (Invitrogen). Libraries were denatured with NaOH according to the manufacturer’s protocol (Ilumina). Sequencing was performed using the MiSeq Reagent Kit V3 with the Ilumina Miseq System. A 10% PhiX internal control (Ilumina) was included in each low-diversity library run.
Sodium hydroxide (naoh)
Manufactured by Illumina
Sourced in United States
NaOH is a chemical compound that serves as a laboratory reagent. It is a white, odorless, and highly corrosive solid. NaOH is commonly used as a pH adjuster, a neutralizing agent, and a cleaning solution in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Miseq reagent kit v3, by Illumina (1 mentions)
Nextera xt index kit v2, by Illumina (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qiaxcel advanced system, by Qiagen (1 mentions)
Ht1 hybridization buffer, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
3 protocols using sodium hydroxide (naoh)
1
16S rRNA Gene Sequencing with Nextera XT
2
Library Preparation and Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA insert fragments in the library were tested using the Agilent 2100 Bioanalyzer (Agilent 1000 Reagents; Agilent Technologies, Inc., Santa Clara, CA, USA), and the concentration was quantified using an ABI StepOnePlus Real-Time PCR System (TaqMan Probe; Applied Biosystems; Thermo Fisher Scientific, Inc.). The aforementioned library was qualified by NaOH according to the protocol (Illumina, Inc., San Diego, CA, USA), diluted with hybridization buffer HT1 (Illumina, Inc.) to a final dilution of 10 pm, then subjected to flowcell hybridization (15 min at 95°C, 30 cycles of 30 sec at 94°C, 30 sec at 59°C and 1 min at 72°C, followed by a final extension cycle of 10 min at 72°C) and then combined with TruSeq PE Cluster kit v3-cBot-HS reagent (Illumina, Inc.) to complete bridge PCR amplification, according to the manufacturer's protocol. Finally, the prepared flowcell was tested using the MiSeq sequencing.
3
Sequencing of IgG Repertoire
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pooled samples were denatured with NaOH according to the protocol (Illumina), diluted with hybridization buffer HT1 to a final dilution of 10 pM, spiked with 5% of PhiX control library and loaded into a 500 cycle version 2 reagent cartridge. Custom primers IgGcSeq and IgGcInd for the read 2 and the indexing read, respectively, were diluted to 0.5 uM in hybridization buffer HT1 and 600 ul loaded into well 19 (index read 1, IgGcInd) and well 20 (read 2, IgGcSeq) of the reagent cartridge. The sample sheet was adapted manually to allow any sequence (N12) as custom index 1. Sequencing was performed for 2 * 250 cycles. The workflow was set to “GenerateFASTQ”. The raw sequencing data have been uploaded to zenodo (doi:10.5281/zenodo.10863).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!