Ultra 2

Manufactured by New England Biolabs

The Ultra II is a high-performance laboratory centrifuge designed for a variety of applications. It features a powerful motor, precise speed control, and a robust construction that ensures reliable and consistent performance. The centrifuge is capable of achieving high rotational speeds, making it suitable for a wide range of sample processing tasks.

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Lab products found in correlation

Nebnext multiplex oligos, by New England Biolabs (1 mentions) 2100 bioanalyzer rna 6000 pico kit, by Agilent Technologies (1 mentions) Directional rna library prep kit, by New England Biolabs (1 mentions) Nebnext rrna depletion kit, by New England Biolabs (1 mentions) Nextseq 500, by Illumina (1 mentions) Epitect bisulphite conversion kit, by Qiagen (1 mentions) Novaseq, by Illumina (1 mentions)

Close spelling variants of this product

NEB Next® Ultra™ II FS DNA PCR-free Library Prep Kit NEBNext Ultra II DNA Library Prep NEBNext Ultra II Directional RNA library preparation kit Next Ultra II DNA Library Prep Kit Ultra Directional RNA Library Prep II NEBNext Ultra II Q5 PCR Master Mix Ultra II FS enzyme Ultra II FS kit NEBNext Ultra II Directional RNA library kit NEBNext Ultra II RNA

3 protocols using ultra 2

Nanopore Library Preparation and Sequencing

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The sequencing library preparation was performed using SQK-LSK108 1D ligation genomic DNA kit following the procedures described below. For each DNA sample, 1.5–2.0 μg of DNA recovered from agarose gel purification was used.

End-repair and dA-tailing were performed using the NEBNext Ultra II End-Repair/dA-tailing Module. In detail, 7 μL of Ultra II End-Prep buffer, 3 μL of Ultra II End-Prep enzyme mix, 5 μL of NFW, and ~ 1.2 μg of DNA were mixed and incubated at 20 °C for 20 min at first followed by another incubation at 65 °C for 15 min.

Sixty microliters of AMPure XP beads was used for purification.

Ligation was performed by adding 20 μL of Adaptor Mix and 50 μL of Blunt/TA Ligation Master Mix to the 30 μL of dA-tailed DNA and then incubation was performed at room temperature for 15 min.

Another purification for the removal of remaining adapters from the adapter-ligated DNA was conducted using 40 μL of AMPure XP beads and ABB buffer supplied in the kit. The purified-ligated DNA was resuspended using 25 μL of ELB, and then the concentration was measured by Qubit to ensure ≥ 500 ng of DNA was retained.

Finally, MinION sequencing was performed using R9.4 flow cells (FLO-MIN 106). A total of eleven independent MinION runs were conducted for all the samples.

Che Y., Xia Y., Liu L., Li A.D., Yang Y, & Zhang T. (2019). Mobile antibiotic resistome in wastewater treatment plants revealed by Nanopore metagenomic sequencing. Microbiome, 7, 44.

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Simultaneous RNA-seq profiling of mouse ESCs and Drosophila SG4 cells

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For cnRNA-seq, 2x10⁷ mouse ESCs (untreated or treated with auxin or dTAG-13 compound for indicated times) were mixed with 8x10⁶Drosophila SG4 cells in PBS. Nuclei were released in 1 mL HS Lysis buffer (50 mM KCl, 10 mM MgSO₄.7H₂0, 5 mM HEPES, 0.05% NP40, 1 mM PMSF, 3 mM DTT, 1x PIC) for 1 min at room temperature, and recovered by centrifugation at 1000 g for 5 min at 4°C, followed by three washes with ice-cold RSB buffer (10 mM NaCl, 10 mM Tris pH 7.4, 3 mM MgCl₂). Pelleted nuclei were resuspended in 1 mL of TRIzol reagent and RNA was extracted according to the manufacturer’s protocol, followed by treatment with the TURBO DNA-free Kit. Quality of RNA was assessed using 2100 Bioanalyzer RNA 6000 Pico kit (Agilent). Next, rRNA was depleted using the NEBNext rRNA Depletion kit (NEB). RNA-seq libraries were prepared using the NEBNext Ultra (for PRC1^deg) or Ultra II (for PRC2^deg) Directional RNA Library Prep kit (NEB), indexed using NEBNext Multiplex Oligos (NEB), polled and sequenced as 80 bp (PRC1^deg) or 40 bp (PRC2^deg) paired-end reads on the Illumina NextSeq 500 in biological triplicates.

Dobrinić P., Szczurek A.T, & Klose R.J. (2021). PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency. Nature structural & molecular biology, 28(10), 811-824.

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Whole-Genome Bisulfite Sequencing for TKO Cells

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Whole-genome bisulphite sequencing for TKO cells expressing DNMT3A and DNMT3B was performed as described (16 (link)). In brief, 6 ug sonicated, genomic DNA were pre-mixed with SssI-methylated phage T7 and unmethylated phage Lambda DNA (both 10 ng, sonicated) and sequencing libraries were prepared using the NEB ULTRA and ULTRAII kits following manufacturer's instructions for genomic DNA library construction and using methylated adaptors (NEB E7535S). Adaptor-ligated DNA was converted by sodium bisulphite using the Qiagen Epitect bisulphite conversion kit. Converted libraries were enriched by 10 cycles of PCR with the following reaction composition: 1 μl Pfu TurboCx Hotstart DNA polymerase (Stratagene), 5 μl PfuTurbo Cx reaction buffer, 25 μM dNTPs, 1 μl universal and 1 μl index primer. PCR cycling parameters were: 95°C for 2 min, 98°C for 30 s, then 10 cycles of 98°C for 15 s, 65°C for 30 s and 72°C for 3 min, ending with one 72°C for 5 min step. The reaction products were purified twice using Ampure-XT beads. Quality of the libraries and size distribution were assessed on an Agilent High Sensitivity tape station. Libraries were sequenced on Illumina HiSeq and NovaSeq machines. All newly generated datasets (Supplementary Table S1) have been deposited to Gene Expression Omnibus (GEO), under accession number: GSE151992.

Mallona I., Ilie I.M., Karemaker I.D., Butz S., Manzo M., Caflisch A, & Baubec T. (2020). Flanking sequence preference modulates de novo DNA methylation in the mouse genome. Nucleic Acids Research, 49(1), 145-157.

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