Sample Preperation

Overview

Good sample preparation is arguably the most critical step in any analytical process because most techniques are unable to detect analytes in situ or results can be distorted by interfering species.  Therefore, finding the right approach to sample preparation is one of the most important, steps to achieving accurate and reproducible results.  Below, we review several forms of sample preparation, solutions for all of which are available through Stratech.

• Tissue Disruption
• DNA & Protein Purification Kits
• Tools for Exosome Research
• Isolate

Suppliers

• Affymetrix USB
• BioSpec
• Cell Crusher
• Systems Bioscience (SBI)

Tissue Disruption

Mechanical disruption is generally needed to access the cellular contents of tough-to-disrupt tissues. Conventionally this is accomplished with a tissue pulveriser, pestle and mortar or bead mill. Stratech supply equipment that makes preparing samples from tougher cells and tissues easy.

Cellcrusher

Cellcrusher (Cellcrusher-CEC) reduces tissues to a fine, easily recoverable powder. Frozen samples are placed in the chilled Cellcrusher and pulverised by striking the device with a mallet. The curved surfaces facilitate effective sample retrieval by means of a contoured recovery spoon and allow cold, dry cleaning, saving time and increasing throughput.

Bead Mill Homogenisers

In bead milling, a large number of minute glass, ceramic or steel beads are vigorously agitated by shaking or stirring. Disruption occurs by the crushing action of the beads as they collide with the cells. By selecting the correct sized beads large and intracellular organelles can often be isolated intact. This is considered the method of choice for disruption for spores, yeast and fungi and works successfully with tough-to-disrupt cells like cyanobacteria, mycobacteria, spores and microalgae. After treatment, the beads settle by gravity in seconds and the cell extract is easily removed. If PCR techniques are to be used, this homogenisation method is one of the few that avoids cross-contamination between samples because both vials and beads are disposable.
Biospec have a range of bead beater homogenisers that vary in the number of samples they are able to process at one time from the MiniBeadBeater-1 for single samples, to the MiniBeadBeater-96, a high throughput machine utilising deep well micro plates, which can process up to 192 samples at a time.
Larger capacity laboratory bead mill cell disrupters agitate the beads with a rotor rather than by shaking. Equipped with efficient cooling jackets, larger sample volumes can be processed without overheating. Biospec’s BeadBeater will disrupt between 15 ml and 250 ml (depending on chamber size) batches of cell suspension in 3-5 minutes.

Stratech supply bead homogenisers of several diffent sizes, from 300 mg scale, to high throughput machines capable of processing up to 192 samples at a time utilising deep-well micro plates, from the Beadbeater (see Fig. 1) up to bead mills that can process 1L of cells.

DNA, RNA & Protein Purification Kits

In molecular and cell biology experiments, sample purity often makes the difference between success and failure. Stratech offer kits for high yield and purity sample preparation that will get your experiments off to a flying start.

From Affymetrix USB® we supply straightforward high recovery (up to 90%) PrepEase® kits for the isolation of plasmids, PCR products, and genomic DNA.
Affymetrix USB®  also offer ExoSAP-IT® single-step PCR cleanup reagent, which utilises Exonuclease I and Shrimp Alkaline Phosphatase to remove unwanted dNTPS and oligonucleotides from PCR reactions.

• 100% product recovery
• Simple, Robot-friendly, one-tube protocol
• Eliminates spin columns, reducing waste & expense
• Fast and scalable for high throughput applications

Precipitation-based genomic DNA purification kits minimise DNA fragmentation that may occur in spin-column or filtration-based methods. Spin column-based kits fragment purified DNA, but have the advantage of speed and convenience versus precipitation-based methods.
Jena Biosciences offer a number of spin column-based kits for genomic DNA preparation from Blood/Animal/Plant (PP-213), Bacteria (PP-214) and Yeast (PP-215).
Stratech offers a selection of kits for a variety of sample types from both Affymetrix USB and Jena Biosciences.
Affymetrix USB and Jena Bioscience also offer excellent value for money Nucleotide / Dye Removal Kits (78776 or  PP-216L), an easy way to remove unincorporated dye-labelled, radiolabelled or unlabelled nucleotides from DNA,  the kits are based on spin column buffer exchange technology and guarantee the highest possible recovery rates in a 5- minute procedure.

DNA Purification

Affymetrix USB® offers straightforward high recovery (up to 90%) kits for the isolation of plasmids, PCR products, and genomic DNA.

• PrepEase® DNA Clean-Up Kit (78758) for purifing DNA fragments from enzymatic reactions or for buffer exchange.
• PrepEase Gel Extraction Kit (78756) for DNA purification from agarose gels or PCR reaction mixtures.

These kits can also be used to remove contaminants such as salts, enzymes, nucleotides, labelling reagents, etc. from a DNA sample prior to PCR.

Genomic DNA Purification Kits are designed for convenient and fast isolation of total DNA from a variety of sample sources. Spin column based methods completely remove PCR inhibitors such as divalent cations and proteins resulting in a high purity preparation of genomic DNA suitable for RT-PCR, Southern blot analysis, genotyping & discovery or validation of SNP/SSR markers. Jena Biosciences offer a number of colum-based kits for Blood-Animal-Plant Preparation (PP-213), Bacteria (PP-214) and Yeast (PP-215).
Solution based systems minimise DNA fragmentation that may occur in spin-column / filtration based methods. From both Affymetrix USB and Jena Biosciences, Stratech offers a selection of kits for a variety of sample types.
For a fast and easy solution to removing unincorporated dye-labelled, marker-labelled or unlabelled nucleotides from DNA, Jena Bioscience offer Nucleotide/Dye Removal Kit (PP-216). The simple clean up procedure is based on gel filtration technology and guarantees highest recovery rates.

RNA Purification

Total RNA Purification Kit (PP-210) from Jena BioScience offers rapid, high purity and high yield isolation of total RNA from small amounts of samples including blood, animal and plant tissue, bacteria and viruses. The spin column based method allows complete removal of inhibitors such as divalent cations and proteins prior to downstream applications.

• High-yield and high-quality total RNA isolation
• Silica-membrane based
• Optimised for a wide range of down-stream applications

Protein Purification

Proteins and enzyme activities frequently interfere with the analysis of small molecules in biological samples. Perchloric acid (PCA) precipitation has been extensively used in sample preparation procedures, as it removes most of the protein present but also stabilises many of the small molecule analytes. Abnova’s Deproteinising Sample Preparation Kit (KA3716) utilises a PCA precipitation method, providing a unique tool to prepare samples for the analysis of a variety of small molecules.

Affymetrix USB range includes highly specific resins for purification of tagged proteins that provide excellent yield and drastically reduced background (PrepEase Protein Purification Kits). These kits may also be used to purify His-tagged proteins from other expression systems such as insect cells, mammalian cells, and yeast. Purification may take place under standard, native or under denaturing conditions, depending on the solubility of the expressed protein.

• Novel – Based on dry silica based resin
• Less non-specific binding of contaminating proteins compared to Ni agarose beads
• Easy to elute protein off column
• Utilizes metal (Ni) affinity gravity-flow chromatography
• High stability against chelating and reducing agents
• Room temperature storage

Tools for Exosome Research

Exosomes are membrane vesicles secreted by most cell types to facilitate intercellular communication. They are found in biological fluids including blood, urine, tumour ascites and tissue culture media, and contain distinct subsets of microRNAs and proteins depending upon the cell type from which they are secreted. This makes exosomes extremely useful for biomarker discovery. Stratech is proud to supply the widest range of market-leading exosome research tools available:

• Isolate: Rapidly isolate bulk exosomes from a wide range of biological fluids without time-consuming ultracentrifugation with ExoQuick and ExoQuick-TC kits, or immunopurify  using specific surface antigens.
• Assay:  Highly sensitive and specific detection and quantitation assay kits using FACS, Western blot, and Exo-ELISA kits.
• Analyse:  Purify and amplify exoRNAs for microRNA qPCR array or microarray studies using a ­­rapid one-day protocol.
• Discover: Identify novel exosome-associated RNA biomarkers using SBI’s complete exosome RNA next-gen sequencing solutions.