Isolation Buffer Research Articles

High-molecular-weight DNA extraction for broadleaved and conifer tree species

Abstract Plant genome sequencing based on long reads has increasingly been applied also to tree species in recent years. A crucial step in these genome projects is the successful extraction of high-molecular-weight DNA in high quality and sufficient quantity, which is imperative for long-read sequencing. The extraction of high-molecular-weight DNA in trees has been limited and difficult to conduct. To achieve successful extraction, modification of the protocol for tree species is necessary. Here, we present a protocol for high-molecular-weight DNA extraction from the broadleaved tree species Fraxinus excelsior and from the conifer species Taxus baccata. The protocol is based on nuclei isolation and is divided into two main steps, i.e. nuclei separation based on the nuclei isolation buffer and DNA extraction using the Nanobind® plant nuclei kit by Pacific Biosciences (PacBio). The protocol can be applied to different tree species to obtain high-molecular-weight DNA in high quality, which can be used for Oxford Nanopore Technologies (ONT) and PacBio sequencing. ONT sequencing using four high-molecular-weight DNA preparations from Fraxinus excel-sior resulted in different read length N50 values (12.91-38.19 kb) and total base output (5.81-23.17 Gb), emphasizing the complex nature of the sequencing pipeline of high-molecular-weight DNA. PacBio HiFi sequencing of Taxus baccata produced circular consensus sequencing reads with an average length of 12.9 kb and an N50 of 13.59 kb. Altogether, this study presents a protocol to obtain high-molecular-weight DNA from different challenging tree species and discusses several important points that can be considered when adapting the protocol to additional species.

Chromosome Analysis and Sorting Using Conventional Flow Cytometers.

The fluorescent dyes Hoechst (HO) and Chromomycin A3 (CA3) are commonly used for bivariate flow karyotyping to distinguish individual chromosomes from one another based on differences in base composition and DNA content. However, analysis of chromosomes using this fluorescent dye combination requires a flow cytometer equipped with lasers of specific wavelengths and higher power than is typical of conventional flow cytometers. This unit presents a chromosome staining technique with a dye combination of DAPI and propidium iodide (PI). Chromosomes stained using this dye combination can be analyzed on conventional flow cytometers equipped with a typical configuration of lasers and optics. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cell culture and metaphase harvest of suspension cell line Alternate Protocol 1: Cell culture and metaphase harvest of adherent cell line Basic Protocol 2: Preparation of chromosome suspension using polyamine isolation buffer Basic Protocol 3: Staining chromosomes with DAPI and propidium iodide Alternate Protocol 2: Staining chromosomes with Hoechst and Chromomycin A3 Basic Protocol 4: Bivariate flow karyotyping on a cell analyzer Basic Protocol 5: Bivariate flow karyotyping on a cell sorter Basic Protocol 6: Purification of flow-sorted chromosomes.

A simple and effective method to purify and activate T cells for successful generation of chimeric antigen receptor T (CAR-T) cells from patients with high monocyte count

BackgroundChimeric antigen receptor T (CAR-T) cells are genetically modified T cells with redirected specificity and potent T-cell-mediated cytotoxicity toward malignant cells. Despite several CAR-T products being approved and commercialized in the USA, Europe, and China, CAR-T products still require additional optimization to ensure reproducible and cost-effective manufacture. Here, we investigated the critical parameters in the CD3+ T-cell isolation process that significantly impacted CAR-T manufacturing's success.MethodsCAR-T cells were prepared from cryopreserved peripheral blood mononuclear cells (PBMC). The thawed PBMC was rested overnight before the CD3+ T cell isolation process using CTS™ Dynabeads™ CD3/CD28. Different isolation media, cell-bead co-incubation time, and cell density were examined in this study. Activated CD3+ T cells were transduced with a gamma retroviral vector carrying the CD19 or BCMA CAR sequence. The CAR-T cells proliferated in a culture medium supplemented with interleukin 2 (IL-2).ResultsCD14+ monocytes hindered T-cell isolation when X-VIVO 15 basic medium was used as the selection buffer. The activation of T cells was blocked because monocytes actively engulfed CD3/28 beads. In contrast, when DPBS was the selection medium, the T-cell isolation and activation were no longer blocked, even in patients whose PBMC contained abnormally high CD14+ monocytes and a low level of CD3+ T cells.ConclusionsIn this study, we discovered that selecting CD3+ T-cell isolation media is critical for improving T-cell activation, transduction, and CAR-T proliferation. Using DPBS as a CD3+ T cell isolation buffer significantly improved the success rate and shortened the duration of CAR-T production. The optimized process has been successfully applied in our ongoing clinical trials.Trial registration NCT03798509: Human CD19 Targeted T Cells Injection Therapy for Relapsed and Refractory CD19-positive Leukemia. Date of registration: January 10, 2019. NCT03720457: Human CD19 Targeted T Cells Injection (CD19 CAR-T) Therapy for Relapsed and Refractory CD19-positive Lymphoma. Date of registration: October 25, 2018. NCT04003168: Human BCMA Targeted T Cells Injection Therapy for BCMA-positive Relapsed/Refractory Multiple Myeloma. Date of registration: July 1, 2019

Systematic Methods for Isolating High Purity Nuclei from Ten Important Plants for Omics Interrogation

Recent advances in developmental biology have been made possible by using multi-omic studies at single cell resolution. However, progress in plants has been slowed, owing to the tremendous difficulty in protoplast isolation from most plant tissues and/or oversize protoplasts during flow cytometry purification. Surprisingly, rapid innovations in nucleus research have shed light on plant studies in single cell resolution, which necessitates high quality and efficient nucleus isolation. Herein, we present efficient nuclei isolation protocols from the leaves of ten important plants including Arabidopsis, rice, maize, tomato, soybean, banana, grape, citrus, apple, and litchi. We provide a detailed procedure for nucleus isolation, flow cytometry purification, and absolute nucleus number quantification. The nucleus isolation buffer formula of the ten plants tested was optimized, and the results indicated a high nuclei yield. Microscope observations revealed high purity after flow cytometry sorting, and the DNA and RNA quality extract from isolated nuclei were monitored by using the nuclei in cell division cycle and single nucleus RNA sequencing (snRNA-seq) studies, with detailed procedures provided. The findings indicated that nucleus yield and quality meet the requirements of snRNA-seq, cell division cycle, and likely other omic studies. The protocol outlined here makes it feasible to perform plant omic studies at single cell resolution.

Ellman's reagent prevents dephosphorylation of histones during isolation of mitotic chromosomes.

Histones H1 and H3 are highly phosphorylated in mitotic HeLa cells but are rapidly dephosphorylated by endogenous protein phosphatases during the isolation of metaphase chromosomes. We show that this dephosphorylation can be prevented by including the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoate) (Ellman's reagent, or DTNB) in the isolation buffer. The minimal amount of DTNB required is approximately stoichiometric with the number of sulfhydryl groups in the lysate. Inhibition of the protein phosphatases can subsequently be reversed by treatment with dithiothreitol or 2-mercaptoethanol. DTNB is compatible with the isolation of either metaphase chromosome clusters or individual metaphase chromosomes. It should be useful in investigations of the structure and biochemistry ofchromatin and chromosomes and in the study of possible functions for mitotic histone phosphorylation.

A low jitter sub‐sampling phase‐locked loop with sampling thermal noise cancellation technique

AbstractThe traditional sub‐sampling phase‐locked loop faces the tradeoffs between phase noise and spur, in that low in‐band phase noise requires large sampling capacitor size but at the sacrifice of spur performance. This paper presents a sub‐sampling PLL aimed at minimizing in‐band phase noise via sampling thermal noise cancellation technique. It enables the substantial reduction of in‐band phase noise while reducing the sampling capacitor size. In addition, due to the reduction of the sampling capacitance, the reference spur performance of the PLL is improved, and the power consumption of the isolation buffer is reduced. Implemented in a 65 nm CMOS process, the in‐band phase noise at 200 kHz offset is −133.4 dBc/Hz at 2.2 GHz and integrated jitter is 80 fsrms. The reference spur is −67 dBc. It consumes 5.5 mA from 1.2 V supply and occupies 0.72 mm2.

Inactivation efficacy of H5N1 avian influenza virus by commonly used sample preparation reagents for safe laboratory practices

The objective of this study was to determine the inactivation efficiency of common sample preparation reagents against highly pathogenic avian influenza A (HPAI) H5N1 virus. HPAI H5N1 virus has caused infections in humans with a mortality rate of over 50%. Due to the high mortality and the risk of aerosol transmission of that virus to humans and birds, infectious HPAI H5N1 viruses are contained in a biosafety level 3 laboratory. However, many procedures for further molecular analyses would be easier in lower biosafety conditions. To ensure the laboratory safety the successful inactivation procedures should be demonstrated before the samples are transferred to a lower containment facility. We tested the inactivation capacity of commonly used cell lysis buffer radio-immuno precipitation assay (RIPA) buffer for protein samples, cell fixatives methanol (MeOH) and paraformaldehyde (PFA) and guanidine isothiocyanate-containing lysis buffer for RNA isolation (RLT, Qiagen) in H5N1-infected cells. Based on our results RLT buffer, 90% MeOH (20 min, −20 °C) and 4% PFA (30 min, RT) all completely inactivated the HPAI H5N1 virus. However, RIPA buffer alone was not sufficient to inactivate the HPAI H5N1 virus in infected cell samples but, instead, combining RIPA lysis buffer and boiling for 10 min the samples in Laemmli buffer led to complete inactivation of the virus.

Integrated Hydraulics-Modeling Platform Automates Managed-Pressure Drilling

This article, written by JPT Technology Editor Chris Carpenter, contains highlights of paper IPTC 19671, “Integrated Hydraulics Modeling for Managed-Pressure Drilling,” by Sayamik A. Rostami, Jose Brana, and Thomas Koithan, SPE, Weatherford. The paper has not been peer reviewed. Copyright 2021 International Petroleum Technology Conference. Reproduced by permission. Technologies aimed at better performance and lower cost are more important to the oil and gas industry than ever. This emphasis drives hardware manufacturers to innovate quickly and create devices with more capabilities so that what was impossible a few short years ago is reality today. In the complete paper, the authors describe a platform that integrates advanced data analytics and hydraulic modeling in real time, coupled with cybersecurity features applied through an open-platform communications unified-architecture protocol directed at automation controllers for managed-pressure-drilling (MPD) applications. MPD Control System An intelligent MPD system automatically controls all the components of an MPD system, including a rotating control device, annular isolation device, flow spool, buffer manifold, choke manifold, and pressure-relief valves. Aside from communicating with and controlling this equipment, such a system is able to communicate with the overarching rig-control system. The system uses the control capabilities of a centralized programmable automation controller with an integrated human/machine-interface screen. Another advantage of the intelligent MPD system is the built-in functionality to track the performance of the system in terms of performance and health through component monitoring to enable condition-based maintenance (CBM).

Multichannel Time Synchronization Based on PTP through a High Voltage Isolation Buffer Network Interface for Thick-GEM Detectors

Data logging and complex algorithm implementations acting on multichannel systems with independent devices require the use of time synchronization. In the case of Gas Electron Multipliers (GEM) and Thick-GEM (THGEM) detectors, the biasing potential can be generated at the detector level via DC to DC converters operating at floating voltage. In this case, high voltage isolation buffers may be used to allow communication between the different channels. However, their use add non-negligible delays in the transmission channel, complicating the synchronization. Implementation of a simplified precise time protocol is presented for handling the synchronization on the Field Programmable Gate Array (FPGA) side of a Xilinx SoC Zynq ZC7Z030. The synchronization is done through a high voltage isolated bidirectional network interface built on a custom board attached to a commercial CIAA_ACC carrier. The results of the synchronization are shown through oscilloscope captures measuring the time drift over long periods of time, achieving synchronization in the order of nanoseconds.

A Low-Jitter and Low-Spur Charge-Sampling PLL

This article presents a low-jitter and low-spur charge-sampling phase-locked loop (CSPLL). A charge-domain sub-sampling phase detector is introduced to achieve a high phase-detection gain and to reduce the PLL in-band phase noise. Even without employing any power-hungry isolation buffers, the proposed phase detector dramatically suppresses the reference spurs by both minimizing the modulated capacitance seen by the voltage-controlled oscillator (VCO) tank and by reducing the duty cycle of the sampling clock. A 50- <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu \text{W}$ </tex-math></inline-formula> RF-dividerless frequency-tracking loop is also introduced to lock the CSPLL robustly when the VCO faces a sudden frequency disturbance. Fabricated in a 40-nm CMOS process, the prototype CSPLL occupies a core area of 0.13 mm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> and synthesizes 9.6-to-12-GHz tones using a 100-MHz reference. At 11.2 GHz, it achieves a reference spur of −77.3 dBc and an RMS jitter of 48.6 fs while consuming 5 mW.

English

Clinacanthus nutans is an economically important medicinal plant that can be found grown in many countries in the Asian region. Useful medicine properties such as anti-cancer, anti-bacteria, and anti-viral, backed by its high content of phytochemical compounds such vitexin, isovitexin, stigmasterol and lupeol has increased the demand for C. nutans in the market. Extensive work had been carried out on its content and pharmacological activity using fresh samples but limited studies for in vitro cultures of C. nutans. No genome size estimation has been done for C. nutans. Objective of this study was to analyse nuclear DNA content of C. nutans using flow cytometry. Preparation of different nuclei isolation buffer and stoichiometric DNA staining using propidium iodide was carried out. The genome size of C. nutans was estimated using Glycine max cv. Polanka as internal standard and its genome size was compared with in vitro plantlets of C. nutans. Flow cytometry analysis revealed that nuclear 2C DNA of C. nutans content is estimated at 1.75 &plusmn; 0.006 pg. Coefficient of variation in flow cytometric analysis was within the limit of 5% implying that the results were reliable with the Tris.MgCl2 being the best nuclei isolation buffer. No significant difference was observed from field grown and in vitro C. nutans. This finding will assist further in genome size evolution analysis of Clinacanthus spp. and to determine polyploids for increased active compounds and biomass. Key word: Flow cytometry; Nuclear DNA content, Clinacanthus nutans.

AE Buffer for RNA Isolation

AE Buffer for RNA Isolation

Using alternative buffer for DNA genomic isolation in forest trees

DNA optimization procedures can be carried out on the type of buffer used during extraction or physical handling techniques in separating genomic DNA from other compounds. The research using Three types of buffer: 1. CTAB, 2. Detergents containing Alkyl Benzene Sulfonate (ABS) surfactants and Three Detergents containing Sodium Laureth Sulphate (SLS) surfactants. This study aims to obtain an optimal DNA isolation method to produce genomic DNA of good quality and sufficient quantity so that it can be used for genetic diversity analysis in forest trees and to determine the optimal alternative buffer for CTAB to facilitate DNA isolation in remote areas, which generally hard to get CTAB. The parameters measured in this study were the presence of DNA and DNA concentration. The results showed that DNA isolation of 12 species of forest trees was successfully carried out using CTAB buffer and Detergent containing ABS surfactants by visualizing the genomic DNA bands from the results of the electrophoresis and Nanodrop spectrophotometer meanwhile Detergent containing SLS surfactants buffer was not successful in DNA isolation. The highest DNA quantity (DNA concentration) was found in 19 samples using CTAB buffer and the detergent containing ABS surfactants buffer with a concentration of 1403,8 - 3412,7 ng/µl. The conclusion of this study was CTAB buffer and the Detergent containing ABS surfactants can also be used as an alternative to a simple buffer for DNA isolation experiments.

A simple flow cytometry-based assay to study global methylation levels in onion, a non-model species.

The online version contains supplementary material available at 10.1007/s12298-021-01047-6.

Isolation of plant nuclei for estimation of nuclear DNA content: Overview and best practices.

A critical aspect for obtaining accurate, reliable, and high-resolution estimates of nuclear DNA content is the release of nuclei from the cytoplasm in sufficient amounts, while maintaining their integrity throughout the analysis, protecting their DNA from degradation by endonucleases, and enabling stoichiometric DNA staining. In embryophytes, the most common method consists of chopping the plant material with a sharp razor blade to release nuclei into an isolation buffer, filtering the homogenate, and staining the nuclei in buffered suspension with a fluorochrome of choice. Despite the recent description of alternative approaches to isolate nuclei, the chopping procedure remains the most widely adopted method, due to its simplicity, rapidity, and effectiveness. In this review article, we discuss the specifics of nuclei isolation buffers and the distorting effects that secondary metabolites may have in nuclear suspensions and how to test them. We also present alternatives to the chopping procedure, options for filtering and fluorochromes, and discuss the applications of these varied approaches. A summary of the best practices regarding the isolation of plant nuclei for the estimation of nuclear DNA content is also provided.

Social buffering of cortisol release and tympanic temperature asymmetries during novelty and isolation stress in marmoset monkeys

Novel environments induce a conflicting emotional approach-withdrawal state that triggers stress-related reactions. Social support through the presence of a highly familiar conspecific buffers the individual against such challenges. Although aversive events seem to be predominantly processed by the right hemisphere, this is still under debate and little is known about functional cerebral asymmetries in nonhuman primates during novelty stress, isolation and social buffering. Here we isolated adult marmoset monkeys in a new open-field arena or in their familiar home-cages to establish hemisphere activity and whether the pairmate’s presence buffers the response. Monkeys socially isolated in either location had higher circulating cortisol levels than non-isolated marmosets, but different hemisphere activity patterns indicated by changes in baseline tympanic membrane temperatures (TMT). The bilateral increase in the monkeys that were isolated in the unfamiliar location may reflect an approach-withdrawal conflict. The left-sided increase in the home-cage isolation group was negatively related to cortisol release, this being potentially associated with a more proactive/approach-prone temperament. Interestingly, TMT and cortisol were unaltered when the pairmate was present. Thus, positive social interaction reduces the perceived intensity of the threat, alters hemisphere asymmetries and blocks the hormonal response to novelty stress, consistent with a buffering effect.

Isolation of Plant Root Nuclei for Single Cell RNA Sequencing.

The characterization of the transcriptional similarities and differences existing between plant cells and cell types is important to better understand the biology of each cell composing the plant, to reveal new molecular mechanisms controlling gene activity, and to ultimately implement meaningful strategies to enhance plant cell biology. To gain a deeper understanding of the regulation of plant gene activity, the individual transcriptome of each plant cell needs to be established. Until recently, single cell approaches were mostly limited to bulk transcriptomic studies on selected cell types. Accessing specific cell types required the development of labor-intensive strategies. Recently, single cell sequencing strategies were successfully applied on isolated Arabidopsis thaliana root protoplasts. However, this strategy relies on the successful isolation of viable protoplasts upon the optimization of the enzymatic cocktails required to digest the cell wall and on the compatibility of fragile plant protoplasts with the use of microfluidic systems to generate single cell transcriptomic libraries. To overcome these difficulties, we present a simple and fast alternative strategy: the isolation and use of plant nuclei to access meaningful transcriptomic information from plant cells. This protocol was specifically developed to enable the use of the plant nuclei with 10× Genomics' Chromium technology partitions technology. Briefly, the plant nuclei are released from the root by chopping into a nuclei isolation buffer before purification by filtration then nuclei sorting. Upon sorting, the nuclei are resuspended in a low divalent ion buffer compatible with the Chromium technology in order to create single nuclei ribonucleic acid-sequencing libraries (sNucRNA-seq). © 2020 Wiley Periodicals LLC. Basic Protocol 1: Arabidopsis seed sterilization and planting Basic Protocol 2: Nuclei isolation from Arabidopsis roots Basic Protocol 3: Fluorescent-activated nuclei sorting (FANS) purification Support Protocol: Estimation of nuclei density using Countess II automated cell counter Alternate Protocol 1: Proper growth conditions for Medicago truncatula and Sorghum bicolor Alternate Protocol 2: Estimation of nuclei density using sNucRNA-seq technology.

Effect Of Endurance Exercise On Localization Of Myoglobin In Mitochondria In Muscle

PURPOSE: Mitochondria play a principal role for metabolism and have a primary role in regulating respiration in myocytes. Recently, we have shown that the muscle-specific protein myoglobin (Mb) interacts with complex IV to augment mitochondrial respiratory capacity in skeletal muscles. However, the precise mechanism for the Mb-mediated upregulation remains unclear. The present study has focused on effect of the endurance training (eTR) on Mb within the mitochondria. METHODS: Wistar male rats aged 10-week, were subjected to eTR sessions for 4 weeks (25 m/min X 60 min X 5-day/week, n=6). Muscle specimen from the deep portion of m. Gastrocnemius was taken and homogenized. Crude mitochondria were isolated by differential centrifugations and washed with the mitochondrial isolation buffer. The isolated mitochondria were treated with proteinase K (PK), osmotic shock (OS), and SDS (or TriX) in order to digest proteins on the outer membrane and in the intramembrane. The final samples were subjected to SDS-PAGE and immunoblotting using antibodies to localize the proteins in the mitochondria. RESULTS: The eTR increased VDAC-I and COX-IV around +80~130% as compared with non-exercise control (p<0.05)., Mb increased by +50% (p<0.05). Western blotting analysis revealed that the PK digested Tom20, and Tom20 band intensity decreased with the amount of PK used. PK treatment, however, did not affect Mb found in the mitochondrial fraction. Combining treatment with PK, OS and SDS (or TrX) allowed immunoblotting detection of the mitochondrial proteins localized in specific regions of the mitochondria. Mb was detected with either PK or OS treatment. But it cannot be detected with a combined PK+OS treatment, suggesting that Mb associated with the inner membrane (intramembrane side, not matrix side) of the mitochondria. The Mb content inside the mitochondria in eTR rat was similar with that in the control muscles (n.s). CONCLUSION: The present results suggest that Mb in muscle cells localizes both in the cytosol and in the mitochondrial intermembrane space. Although eTR elevates mitochondrial volume and Mb content but does not change Mb content in the mitochondria. Therefore, the observation might imply that the dynamic flux of Mb from cytosol to mitochondria has greater importance than just the amount found in the mitochondria

A simple plant high-molecular-weight DNA extraction method suitable for single-molecule technologies

BackgroundHigh-molecular-weight and pure DNA is crucial for high-quality results from 3rd generation DNA Analyzers and optical mapping technologies. Conventional nuclei isolation methods for preparing high-molecular-weight genomic DNA from plant tissues include the preparation of protoplasts or embedding nuclei in an agarose matrix with subsequent manipulations via electro-elution or pulsed-field gel electrophoresis.ResultsIn this method, plant nuclei are isolated by physically grinding tissues and reconstituting the intact nuclei in a unique Nuclear Isolation Buffer (NIB). The plastid DNAs are released from organelles and eliminated with an osmotic buffer by washing and centrifugation. The purified nuclei are then lysed and further cleaned by organic extraction, and the genomic DNA is precipitated with a high concentration of CTAB. The highly pure, high molecular weight gDNA is extracted from the nuclei, dissolved in a high pH buffer, allowing for stable long-term storage.ConclusionsThis method is unique and avoids the use of embedding in agarose, which dramatically reduces time (4–8 h versus days), complexity, and materials cost. This procedure can be used on essentially any plant species and tissue stage. Here we describe a case study and a simple method to rapidly prepare high molecular weight gDNA from Upland cotton, blackgrass, and strawberry suitable for single-molecule sequencing.

Rooibos (Aspalathus linearis) Genome Size Estimation Using Flow Cytometry and K-Mer Analyses.

Plant genomes provide information on biosynthetic pathways involved in the production of industrially relevant compounds. Genome size estimates are essential for the initiation of genome projects. The genome size of rooibos (Aspalathus linearis species complex) was estimated using DAPI flow cytometry and k-mer analyses. For flow cytometry, a suitable nuclei isolation buffer, plant tissue and a transport medium for rooibos ecotype samples collected from distant locations were identified. When using radicles from commercial rooibos seedlings, Woody Plant Buffer and Vicia faba as an internal standard, the flow cytometry-estimated genome size of rooibos was 1.24 ± 0.01 Gbp. The estimates for eight wild rooibos growth types did not deviate significantly from this value. K-mer analysis was performed using Illumina paired-end sequencing data from one commercial rooibos genotype. For biocomputational estimation of the genome size, four k-mer analysis methods were investigated: A standard formula and three popular programs (BBNorm, GenomeScope, and FindGSE). GenomeScope estimates were strongly affected by parameter settings, specifically CovMax. When using the complete k-mer frequency histogram (up to 9 × 105), the programs did not deviate significantly, estimating an average rooibos genome size of 1.03 ± 0.04 Gbp. Differences between the flow cytometry and biocomputational estimates are discussed.