The bacterial V4 hypervariable region of the 16S ribosomal RNA sequence was PCR amplified from isolated DNA using “universal” 16S primers (515F and 806R). Reverse primers contained a unique 12-base pair Golay barcode that was incorporated into the PCR amplicons (Fierer et al., 2008 (link)). Amplicons were sent to The Scripps Research Institutes NGS core facility for cleaning using DNA Clean & Concentrator™-25 columns (Zymo, Irvine, CA), quantification using a Qubit Fluorometer (Life Technologies, Thermo Fisher Scientific), and pooling. Pooled PCR products were size selected on a 2% agarose gel (290–350bp) and purified using a Zymoclean™ Gel Dna recovery kit to prepare sequencing libraries following the recommended Illumina protocol involving end repair, A-tailing and adapter ligation. The library was then amplified for 12 cycles using the HiFi Polymerase (Kapa Biosystems, Wilmington, MA). The purified DNA library was quantitated and denatured in 0.1 N NaOH and diluted to a final concentration of 5 pM. Sequencing was performed using a 2x300 kit (version 3) on the Illumina MiSeq® system (Illumina, Inc., San Diego, CA, USA) along with 4 pM PhiX control library. Raw sequence reads were submitted to the Sequence Read Archive (SRA; Accession number: PRJNA417868) as an NCBI BioProject (ID: 417868) accessible at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA417868 .
Zymoclean gel dna recovery kit
Manufactured by Illumina
Sourced in United States
The Zymoclean™ Gel DNA Recovery Kit is a laboratory tool designed for the purification of DNA fragments from agarose gels. It is used to extract and concentrate DNA samples that have been separated by gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq genome analyzer, by Illumina (2 mentions)
Miseq system, by Illumina (1 mentions)
Hifi polymerase, by Roche (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Dna clean concentrator 25 column, by Zymo Research (1 mentions)
Dna preparation kit and protocol, by Illumina (1 mentions)
Dna clean and concentrator 5 kit, by Zymo Research (1 mentions)
3 protocols using zymoclean gel dna recovery kit
1
Amplification and Sequencing of Bacterial 16S V4 Region
2
Bisulfite-Sequencing Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EpiQuest libraries were prepared according to manufacturer’s protocol [Zymo
Research (ZR), Irvine, CA, USA] and as previously described.16 (link) Genomic DNA (200–500 ng) was digested with 60 units of
TaqI and 30 units of MspI
(NEB, Ipswich, MA, USA) sequentially. Size-selected
TaqI-MspI fragments (40–120 bp and
120–350 bp) were filled-in and 3′-terminal-A extended, extracted with a ZR DNA
Clean and Concentrator™-5 kit. Ligation to pre-annealed adapters
containing 5′-methyl-cytosine was performed using the Illumina’s DNA preparation
kit and protocol (Illumina, San Diego, CA, USA). Purified adaptor ligated
fragments were bisulfite-treated using the EZ DNA Methylation-Direct™Kit (ZR). Preparative-scale PCR was performed. DNA Clean and
Concentrator™ -purified PCR products were subjected to a final
size selection on a 4% NuSieve 3:1 agarose gel. SYBR-green-stained gel slices
containing adaptor-ligated fragments of 130–210 bp or 210–460 bp in size was
excised. Library material was recovered from the gel (Zymoclean™ Gel
DNA Recovery Kit, ZR) and sequence on an Illumina HiSeq Genome Analyzer.
Research (ZR), Irvine, CA, USA] and as previously described.16 (link) Genomic DNA (200–500 ng) was digested with 60 units of
TaqI and 30 units of MspI
(NEB, Ipswich, MA, USA) sequentially. Size-selected
TaqI-MspI fragments (40–120 bp and
120–350 bp) were filled-in and 3′-terminal-A extended, extracted with a ZR DNA
Clean and Concentrator™-5 kit. Ligation to pre-annealed adapters
containing 5′-methyl-cytosine was performed using the Illumina’s DNA preparation
kit and protocol (Illumina, San Diego, CA, USA). Purified adaptor ligated
fragments were bisulfite-treated using the EZ DNA Methylation-Direct™Kit (ZR). Preparative-scale PCR was performed. DNA Clean and
Concentrator™ -purified PCR products were subjected to a final
size selection on a 4% NuSieve 3:1 agarose gel. SYBR-green-stained gel slices
containing adaptor-ligated fragments of 130–210 bp or 210–460 bp in size was
excised. Library material was recovered from the gel (Zymoclean™ Gel
DNA Recovery Kit, ZR) and sequence on an Illumina HiSeq Genome Analyzer.
3
Genome-wide DNA Methylation Analysis by RRBS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA samples were shipped on ice to Zymo Research (Irvin, CA) for EpiQuest library preparation and genome-wide DNA methylation analysis by reduced representation bisulfite sequencing (RRBS). Briefly, 200–500 ng of genomic DNA were digested first with TaqI followed by digestion with MspI (Ipswich, MA, USA). Size-selected TaqI-MspI fragments (40–120 bp and 120–350 bp) were filled-in and 3′-terminal-A extended, then extracted by Zymo Research DNA Clean and Concentrator-5 kit (Irvin, CA). Ligation to pre-annealed adapters containing 5′-methyl-cytosine was performed using Illumina’s DNA preparation kit and protocol (San Diego, CA). Purified adaptor-ligated fragments were bisulfite-treated using the EZ DNA Methylation-Direct Kit (Irvin, CA). Preparative-scale PCR was performed. DNA Clean and Concentrator-purified PCR products were subjected to a final size selection on a 4% NuSieve 3:1 agarose gel. SYBR green-stained gel slices containing adaptor-ligated fragments of 130–210 bp or 210–460 bp in size were excised. Library material was recovered from the gel (Zymoclean Gel DNARecovery Kit, Irvin, CA, USA) and sequenced on an Illumina HiSeq Genome Analyzer (San Diego, CA).
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!