Tools and Protocols
Sample Preparation
Preparation of high-quality DNA and protein samples from biological material is a critical first step of most biotechnology applications. The CBFG maintains a variety of instruments to aid in sample preparation.
Instruments Used in Sample Preparation
Centrifuges
Assorted centrifuges are available for a wide range of vessels and accompanying rotors, including two plate rotor centrifuges for microplates (both standard and deep well) are is available.
PCR Machines
Quantitative polymerase chain reaction or qPCR, as well as reverse transcriptase qPCR (gene expression) RT-qPCR, are important applications supported by the CBFG. For these experiments, we use:
Corbett Rotor-Gene 3000™
Qiagen Rotor-Gene Q 5-Plex HRMplatform
- Capable of using five fluors (simultaneously) as well as High-Resolution Melt applications (taxa ID, epigenetics, mutation scanning)
- Up to 100 samples concurrently
Bio-Rad CFX Connect (96 well)
Thermo Fisher QuantStudio 7 Flex Real-Time PCR System
- 96 or 384 well sample blocks
- 6 colour multiplexing
- Experimental design is critical to success in qPCR experiments. The CBFG staff provides training and consultation for all aspects of qPCR based on MIQE recommended guidelines, ranging from experimental design and instrument operation to data interpretation and validation to ensure your best results.
Imaging Systems
We have an AlphaInnotechHP imager for nucleic acid, and we have a Bio-Rad ChemiDocMP imager for fluorescence and chemiluminescence detection (four-fold log sensitivity).
epMotion 5073m NGS Liquid Handling Workstation
For automated, high-throughput sample preparation a epMotion 5073m is available. The CBFG staff provides training, consultation, and programming for robotic sample preparations with this system.
Promega Maxwell® 16 System
Generating some of the highest purity nucleic acid isolations, an easy, medium-throughput bead agitation robotic sample preparation Promega Maxwell 16 System is available. This system is ideal for the purification of genomic DNA or RNA from virtually any starting material, is extremely cost-effective, and can process between one and sixteen samples simultaneously.
Spectrophotometers and Fluorospectrometers
We have a UV-VIS Spectrophotometer as well as low volume UV-VIS Nanodrop ND2000 and Fluorescent ND3300 spectrophotometers, in addition to a Qubit4 fluorescent system for the accurate quantification of nucleic acids and/or proteins.
Agilent 2100 Bioanalyzer
Quality control (quantity, size, quality) of DNA, RNA, and Protein samples can be analyzed on our Agilent 2100 Bioanalyzer.
Sage BluePippin
Size-Selection of DNA Fragments (100 bp ~ 50kbp). Accurate, reproducible, high yield recovery of size- selectable DNA fragments. Primarily for use in NGS applications (both short-read (Illumina) and long-read (PacBio)). This instrument significantly improves genome assemblies and transcriptome completeness.
Covaris M220 Ultrasonicator
Can be used for cell lysis: mammalian, bacterial, yeast; cfDNA Extraction from Plasma, DNA Extraction from Dried Blood Spots, DNA/RNA/Chromatin Shearing, DNA/RNA/Total NA/Protein Extraction from FFPE, Tissue lysis, and extraction. Completely user-size selectable fragments using a Peltier-controlled ultrasonication chamber.
DNA Sequence Analysis
The core activity of the CBFG is DNA sequence analysis. It is the order of nucleotide bases (adenine, guanine, cytosine, thymine) in a molecule of DNA that defines every living organism. We are well-equipped to support any sequencing projects, large or small.
Instruments Used in DNA Sequence Analysis
The CBFG houses and maintains a variety of DNA sequencing instruments that can analyze individual samples, one at a time, or many samples at once.
These include:
- Applied Biosystems 3130xl Genetic Analyzer
- Illumina MiSeq Benchtop next-generation sequencer
- Oxford Nanopore minION
The CBFG staff provides training and consultation for all aspects of DNA sequence analysis from sample preparation to data interpretation – especially for the Next Generation Sequencing (NGS). You can also download our Guide to DNA Sequence Analysis and template preparation.
DNA Fragment Analysis
In order to sequence long DNA pieces, such as a whole chromosome (a cell structure consisting of DNA and protein), it is necessary to work with much shorter DNA fragments. The DNA sequencing instrumentation in the CBFG is also used for various types of DNA fragment analysis.
Instruments Used in DNA Fragment Analysis
The DNA sequencing instruments in the CBFG can also be used for several different kinds of DNA fragment analysis. We use:
- Applied Biosystems GeneMapper® software or
- Sequence Scanner Software
for interpretation of fragment analysis data and a variety of programs for phylogenetic analysis.
Real-Time PCR
Real-time PCR (polymerase chain reaction) is perhaps the most important tool for analysis of gene expression. PCR enables us to generate many copies of a single DNA piece. The CBFG maintains several different instrument platforms for real-time (quantitative) PCR and provides training for real-time PCR experimental design.
Instruments Used in Real-Time PCR
Quantitative polymerase chain reaction or qPCR, as well as reverse transcriptase qPCR (gene expression) RT-qPCR, are important applications supported by the CBFG. For these experiments, we use:
- Qiagen Rotor-Gene Q 5-Plex HRM platform
- Bio-Rad CFX Connect (96 well)
- Capable of using five fluors (simultaneously) as well as High-Resolution Melt applications (taxa ID, epigenetics, mutation scanning)
- Up to 100 samples concurrently
- Thermo Fisher QuantStudio 7 Flex Real-Time PCR System
- 96 or 384 well sample blocks
- 6 colour multiplexing
These instruments are calibrated for most of the fluorophores that are commonly used for qPCR experiments.
Experimental design is critical to success in qPCR experiments. The CBFG staff provides training and consultation for all aspects of qPCR based on MIQE recommended guidelines, ranging from experimental design and instrument operation to data interpretation and validation to ensure your best results.
Proteomics
A growing area of support in the center is proteomics, with the addition of a Thermo Fisher OrbiTrap MS in Pearson Hall. Like genomics, which is the study of large sets of genes, proteomics is the study of large sets of proteins and how these proteins change under different biological conditions. The CBFG provides training and equipment for 2-D gel-based proteomics, MALDI MS (Dept of Chemistry) and OrbiTrap MS (Dept of Microbiology).
Instruments Used in Proteomics
Proteomics is a complementary field to nucleic acid investigations supported by the CBFG. We have much of the preparatory equipment and protocols required to perform highly parallel, 2-dimensional gel electrophoresis, as well as prepare samples for mass spectrometry (MALDI and OrbiTrap).
A Bio-Rad ChemiDoc MP Imaging System for Stain-Free Western Blotting. This system can be used for imaging both 1-D and 2-D gels. This system can produce high-quality images with a very wide variety of staining methods including colourmetric, chemiluminescent and fluorescent stains. With four-fold log sensitivity, this is a flexible and versatile instrument.
ÄKTA FPLC (Fast Protein Liquid Chromatography) purification system from Cytiva Life Sciences offers users the ability to purify proteins from complex starting materials such as tissue homogenates. Routine and reproducible purifications with UV detection, dual pump system, a variety of CBFG maintained columns for SEC, Affinity, IEX, HISTrap, and other applications are available. Automatic fraction collection is also available, as is training on the UNICORN software.
Bioinformatics
Bioinformatics is the science of managing DNA and protein sequence data – aka ‘big data’. The CBFG provides a very wide range of software and Linux-based computer resources for bioinformatics.
Instruments Used in Bioinformatics
Bioinformatics is an area that encompasses a wide range of computer resources for the management of DNA and protein sequence information as well as information about biological function. Bioinformatics is at the nexus of "big data" and biology.
The CBFG has a dedicated computer room facility that houses four workstations with
- Linux (Ubuntu)
- Windows10
on which are pre-installed proprietary bioinformatics programs and utilities, including:
In addition to paid software, the CBFG can install & compile, of advise regarding installation of any open-source software that maybe needed for analysis of data. In collaboration with Miami University's RedHawk High Performance Computing Group, we have access to this on-site cluster that contains many bioinformatics analysis programs pre-installed and configured for parallel processing. As part of the Ohio University System, Miami University is permitted to use of the resources of the State-funded Ohio Supercomputer Center.
Training and coordination for all of these resources are available in the CBFG. These bioinformatics tools can also be made available for use by appropriate courses depending on the size of the class.