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Application Notes
Protocols
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Guides
Protocols
CTAB Protocol for Isolating DNA from Plant Tissues
A protocol for isolating DNA from plants using a traditional CTAB method, using chloroform. This protocol can isolated DNA from fresh, frozen, or freeze dried plant samples. Successfuly isolated DNA from
Anthurium
, Corn (
Zea mays
), Cotton, Grape, Pine Needles, Rape Seed, Rice, Rye, Sorghum, Soybean, Sunflower, and Wheat
Rapid Field Preparation for Plant DNA Isolation
A simple protocol for preserving the integrity of the DNA until samples can be processed in a lab.
Alternative Protocols for the Isolation of RNA
The Synergy™ nucleic extraction chemistry, originally developed for plant DNA isolation, has successfully been used for total nucleic acid extraction and RNA isolation. For both SYNERGY™ 2.0 and SYNERGY™ 96 well.
Isolation of Soil Microbiome using Modified Synergy™ Protocol
Isolating DNA from the soil microbiome can be challenging. OPS Diagnostics' Synergy™ chemistry originally designed for plants was tested and modified to effectively isolate DNA from soil. Synergy™ incorporates the bead beating step of homogenization into the purification scheme using a proprietary chemistry that binds contaminants to the Synergy™ grinding matrix.
E. coli
Plasmid DNA MiniPrep Protocol
Isolating plasmid DNA from
Escherichia coli
using an alkaline lysis is a well-established method.
Lactate Dehydrogenase Protocol
Lactate dehydrogenase (LDH) is an enzyme which is useful for monitoring cell death and disruption. LDH detection assays are can be used to assess the efficiency of homogenization methods.Here is a protocol for running a LDH assay in house without the use of kits.
Homogenization Application Table
List of protocols for Homogenizing different biological samples, including bacteria, fungi, pills, animal tissue, clay...etc.
Bacteria Freeze Drying Protocol
Freeze drying bacteria (lyophilization) is an established method for the archiving and long-term storage. The following general protocol is aimed at providing a guide for individuals who are new to lyophilizing bacteria.
Protocol of Freezing Bacteria in Glycerol
Bacteria can be preserved by being frozen. The process is simple and requires screw cap microfuge tubes and sterile glycerol.
Storage of Bacterial Cultures at 4°C
Storing bacteria in a refrigerator is practical for short periods of time. having a readily available active culture can be practical.
High Throughput Isolation of Plant DNA using the 96 Well Synergy™ Plant DNA Extraction Kit
A bead beating based kit, allowing for high throughput sample processing. Impurities are captured on the Synergy™ grinding matrix and DNA in the lysate is further purified using filter plates. Tested on
Zea mays
(corn), Duckweed, tobacco, Sorghum, wheat and more.
Cryogenic Homogenization of Animal Tissue
A protocol on cryogenically homogenizing animal tissue for RNA isolation. Specifically for samples, less than 100 mg processed using a CryoGrinder™, which is a miniaturized mortar with a motorized pestle.
Homogenization of Resilient Tissues by Bead Beating
Homogenizing many tissues, presents a unique set of challenges due to the prescenes of elastic connective tissue. Bead beating can effectively homogenize these resilient tissues, as well as bone, and result in a highly fluid homogenate with small particles sizes.
Homogenization of Soft Animal Tissue with a Rotor Stator
Soft animal tissue, or tissue with little connective tissue can be homogenized with the Rotor Stator.
Cryogenic Homogenization of Skin and Sclera
Homogenizing tissue with elastin and collogen requires liquid nitrogen to prevent the samples from becoming rubbery.
Bacterial Bead Beating
General parameters and considerations for disrupting bacterial cells, using bead beating.
Homogenizing Yeast by Bead Beating
General parameters and considerations for disrupting yeast cells using bead beating methods.
High Throughput Disruption of Yeast in a 96-Well Format
A protocol for the mechanical disruption of yeast using 96 well format and a high throughput homogenizer.
Homogenizing Fungi by Bead Beating
General parameters and consideration for disrupting fungal cells. Considerations, are for both low throughput vortexing indivdual tubes to high throughput homogenizers and plates.
Mycobacterium
Cell Disruption Methods
Mycobacterium's
unique outer layer of lipids makes it resilient to most disrupting methods. This is a simple bead beating method to lysis this tough bacteria.
Soybean Homogenization
Homogenizing dried soybean seeds for high throughput extraction, for single seed and pooled seeds processing.
Maize Homogenization
Zea mays
(corn), can be pulverized by employing several methods depending on the part of the plant and the desired end result.
Bead beat a single corn kernel
Bead beat multiple corn kernels pooled
Traditional grinding using liquid nitrogen, with a mortar and pestle
High Throughput homogenization using a 96 well plate
General Plant Homogenization
General protocols and considerations for homogenizing leaves and seeds using bead beating.
Rat Feces Homogenization Protocol
Following collection, the initial step for analysis is to homogenize the sample. High throughput homogenization can speed up sample processing while also containing the samples, thus reducing the risk of exposure.
Pulverizing of Tablets and Pills
Pills and tablets are homogenized dry using stainless steel grinding balls. The resulting product is a very fine powder.
Fingernail Homogenization using a High Throughput Homogenizer
Fingernails can easily be homogenized using a high throughput homogenizer and a stainless steel grinding ball. This is a standard protocol for fingernail homogenization, using human nails.
Videos
Microbial Freeze Drying (webinar)
A recorded webinar on how microbial freeze drying works, as well as the different methods and considerations for successful freeze drying of bacteria.
High Throughput Homogenization of Plants (webinar)
A recorded webinar on the tricks and tools used for high throughput processing of plants, focusing on plate homogenizers.
Synergy™ Plant DNA Extraction Kit (video)
This video demonstrates the novel chemistry used in isolating DNA from plants, using the Synergy™ Plant DNA Extraction Kit.
CryoCooler™ (video)
A video demonstration on how to use the Cryocooler™, for different labaoratory processes.
CryoGrinder™ (video)
A video demostration on how to use the CryoGrinder™, to homogenize samples.
Cryogenic Grinding of Rat Skin (video)
Homogenizing skin presents a unique set of challenges, this is a demonstration of how to homogenize rat skin using the CryoGrinder™.
Bead Beater Homogenizers (video)
There are so many homogenizers to choose from, here is a video comparison of the homogenizers offered by OPS Diagnostics.
AC Block™ (video)
The AC Block™ is used to keep temperature sensitive samples below -130°C while homogenizing. This video gives an overview of its use.
Guides
Guide to the Disruption of Biological Samples - 2012
Disruption is an early and fundamental step in any research which involves analyzing, separating, or isolating some component from an intact sample. This includes the isolation/harvesting of cellular components or quantification of nucleic acids (i.e. DNA and RNA), proteins, and analytes.
Considerations in Formulating Freeze Dried (Lyophilized) Quality Control Cultures
Freeze drying can be extremely useful for producing quality control samples that have unique and targeted specifications. However doing so requires an understanding of the parameters that impact the stability of microbes.
Sample Homogenization Considerations for NextGen Sequencing
The note looks at sample preparation methods and the impact they can have on DNA used in Next-gen sequencing. As well as offering tips for optimizing the preparation protocol for different downstream sequencing methods.
Bead Beating: A Primer
A guide to homogenize biological samples via bead beating. The guide outline is effective mechanical method used on a wide range of biological samples including microorganisms, plants, and animals.
A Comparison of Grinding Media Composition
The grinding media in the tube can have a great impact on sample preparation. To help determine the best grinding media for different applications. This guide outlines the different physical properties of grinding media.
Selection of Grinding Vials for Sample Processing
Reference guide for selecting grinding vials, based on sample type and machine. Grinding Vials Differentiation and Sample Processing Application Table
Comparison of Pre-Filled Disruption Tubes
A table comparing OPS Diagnostics' Prefilled tubes against two competitors tubes. As well as a reference table for choosing the right prefilled tube for different biological samples.
Tools for Bead Beating
An overview of the tools needed to efficiently bead beat biological samples. Including vials, balls/beads, and homogenizers.
The Effect of Bead Beating on RNA Integrity
Investigation into the effects of bead beating RNA quality using OPS Diagnostics’ bead beating processes and a commercial RNA isolation kit. RIN was used to assess the integrity of the samples.
Co-Isolation of Plant and Pathogen/Parasite DNA and Its Potential Use in Disease Detection and Monitoring.
Exploring the ability of the Synergy™ 2.0 Plant DNA Extraction Kit, to rapidly assess the prescences of pathogen, for real world application.
Cryogenic Bead Beating (excerpt from "Bead Beating: A Primer")
Overview on using Cryoblock™ for cryogenic grinding.
Reduction of Nonspecific Binding of Yeast Protein to Grinding Media
To reduce nonspecific binding during homogenization, OPS Diagnostics developed Low Binding grinding beads that undergo a proprietary process to minimize nonspecific binding of molecules. A demonstration the effectiveness of Low Binding grinding beads, yeast was used as a test for homogenization.
Zirconium Beads for the Isolation of DNA from
Mycobacterium
Low binding beads help to provide greater sensitivity in detecting bacteria when the number of microbes in the original sample is low. Modifying the beads with a low-binding surface helps to minimize nucleic acid loss due to non-specific adsorption. Furthermore, the beads themselves provide a surface for non-specific adsorption of nucleic acids.
Impact of Method Variables on Microbiota Sample Disruption
When measuring microbial diversity in samples, whether fecal, soil, or sediments, obtaining ribosomal RNA genes that are representative of the population is critical. The laboratories at OPS Diagnostics have analyzed variations associated with sample processing and found the overall method, equipment applied, and choice of accessory items can all dramatically affect data.
Evaluation of the Synergy™ Rapid Plant DNA Isolation Chemistry
Synergy™ combines bead beating with an extraction matrix that disrupts the sample and captures contaminates. Evaluates this novel chemistry with two traditional isolation methods.
Addendum: qPCR of DNA Isolated with CTAB, DNeasy®, and Synergy™ Methods
A follow up to "Evaluation of the Synergy™ Rapid Plant DNA Isolation Chemistry", this note supplements the previous by examining plant DNA isolation with qPCR. Determining the relative effect of different isolation methods on the quanitity of DNA recovered.
Two-Step Purification Strategy using Synergy™ Chemistry and Silica Spin Columns Improves Yield and Quality of Plant DNA.
A product note on the incorporation of silica spin columns into the Synergy™ protocol. As well as present a comparison of both Synergy™ protocols to a commerically available kit.
Comparison of Three CTAB Buffers for Plant DNA Isolation
This study makes a comparison between three different CTAB plant DNA extraction buffers. One is a homegrown recipe, while the other two are commerically available. The buffers were compared by isolating DNA from two week old corn plants.
Comparison of Methods for DNA Extraction from Soybeans
This analysis demonstrates that soybean homogenates generated from the high throughput homogenization are suitable for genetic analysis. Four different DNA methods were compared for the isolation of DNA from the soybean homogenates.
Homogenization Options of Leaf Tissue for Nucleic Acid Isolation
Overview of methods and considerations for extracting nucleic acids (DNA and RNA) from plants. Including cryogenic with mortar and pestle, wet with a rotor stator, and bead beating.
Helpful Tools for Isolating DNA from Cereal Crops
An overview of high throughput homogenization of plants leaves and seeds, as well as cryogenic grinding.
A Guide to Bacterial Preservation: Refrigeration, Freezing, and Freeze Drying
In depth analysis of different methods for preserving bacterial samples. Discussing the strengths and weakness of the methods.
Bacteria Lyophilization Overview
Freeze drying bacteria (lyophilization) is a very well established method for the archiving and long-term storage. The general process is used to preserve bacteria, fungi, yeasts, proteins, nucleic acids, and any other molecules which may be degraded due to the presence of water.
Lyophilization Reagents Shelf Life Study
A comparison of retained sample of lyophilization reagent in a functional assay to measure the potency of the formulation over time.
Research Note: In Pursuit of a Ready-To-Use, Shelf-Stable qPCR Mix
Shelf-stable qPCR mixtures have practical applications in field and point-of-care diagnostic testing. Freeze-drying is one technique used to formulate shelf-stable products.
Survival of
Escherichia coli
in Four Lyophilization Solutions
This study looks at the ability of two different commerically available Microbial Freeze Drying Buffers, sucrose, and skim milk to protect
Escherichia coli
following lyophilization.
Considerations in DNA Hybridization Assays
Many factors influence the success of DNA hybridization assays, including the length, composition and sequence of the probe, hybridization buffer composition, temperature of hybridization, concentration of target strand and probe, and whether the assay is solution based or solid phase.
Optimizing Enzyme Immunoassays in Plates
Many parameters can affect the performance of an enzyme immunoassay, unless properly designed and optimized, an enzyme immunoassay may create more questions.
Maintenance of Protein Stability
Maintaining the integrity of proteins structure and function
in vitro
during isolation as well as storage can be challenging. This note outlines aspects of handling and storage which play important roles in the stability of proteins.
Factors Affecting Protein Stability
In Vitro
A follow up to "Maintenance of Protein Stability" this note focuses on the handling of proteins during isolation and the factors that result in a successful extraction of proteins.
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