RFLP Analysis

T-RFLP analysis on samples isolated from two
different time points in a time course study.

T-RFLP analysis is a technique used to study complex microbial communities based on variation in the 16S rRNA gene. T-RFLP is a culture-independent, rapid, sensitive and reproducible method of assessing diversity of complex communities without the need for any genomic sequence information. It has been used to examine bacterial populations in diverse natural habitats and to understand community structure and dynamics in response to changes in environmental or physiological parameters.

Products
Experimental Workflow: Find the Products You Need for Every Step
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Step 1: Extract DNA

DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction. Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction.

Products for Step 1: Extract DNA

NucPrep® DNA Chemistry for Tissues

Starter kit for DNA isolation from animal and plant tissues and cells, etc., using NucPrep® Chemistry.

MagMAX™ Express Magnetic Particle Processor

MagMAX™ Express magnetic particle processors are ideal for researchers who want to automate their MagMAX sample preparation kits. The MagMAX Express platform is the premier automated platform to meet your throughput needs.

MagMAX™ Express-96 Magnetic Particle Processors

MagMAX™ Express magnetic particle processors are ideal for researchers who want to automate their MagMAX sample preparation kits. The MagMAX Express platform is the premier automated platform to meet your throughput needs.

Step 2: PCR

PCR of 16S rRNA fragment with 5’-fluorescently labeled primer pair (e.g. 6-FAM and PET, resp).

Products for Step 2: PCR

5' Fluorescent Labeled Oligo

We offer a portfolio of fluorescent dyes for gene expression studies, 5' nuclease assays, PCR, fragment analysis, and genotyping. When you know the exact sequence of a fluorescent labeled probe or primer, we can synthesize it for you.

Step 3: Restriction Digest

Digest PCR amplicons with restriction enzyme(s).

Step 4: Prepare Samples for Fragment Analysis Run

Add aliquot of PCR / restriction digest reaction to size standard and Hi-Di Formamide mix; heat denature and proceed with electrophoresis.

Products for Step 4: Prepare Samples for Fragment Analysis Run

Hi-Di™ Formamide

Highly deionized formamide used to resuspend samples before electrokinetic injection in capillary electrophoresis systems.

GeneScan™ Size Standards

GeneScan™ Size Standards are fluorescently-labeled DNA fragments required for performing fragment analysis and genotyping applications on our capillary electrophoresis platforms.used to determine the size of unknown fragments in an experimental sample.

Step 5: Electrophoresis

After performing the post-sequencing reaction purification, samples are ready for analysis on an Applied Biosystems capillary electrophoresis-based genetic analyzer. During capillary electrophoresis, the products of the cycle sequencing reaction migrate through capillaries filled with polymer. The negatively charged DNA fragments are size separated as they move through the polymer in the capillaries toward the positive electrode.

Products for Step 5: Electrophoresis

3500 Genetic Analyzer

The 3500 8-capillary platform can run a wide variety of applications—including de novo sequencing and resequencing (mutational profiling)—as well as microsatellite analysis, MLPA™, LOH, MLST, AFLP® and SNP validation or screening. The majority of applications can be run on a single polymer and capillary array.

3500xl Genetic Analyzer

The 3500xL 24-capillary platform can run a wide variety of applications, including de novo sequencing and resequencing (mutational profiling), as well as microsatellite analysis, MLPA™, LOH, MLST, AFLP® and SNP validation or screening. The majority of applications can be run on a single polymer and capillary array.

3730 DNA Analyzer

The 48-capillary 3730 DNA Analyzer is the Gold Standard in medium-to-high throughput genetic analysis. Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. Get the highest quality data at a low cost per sample. This is upgradeable to 96 capillaries.

3730xl DNA Analyzer

The 96-capillary 3730xl DNA Analyzer is the Gold Standard for high throughput genetic analysis. Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. Get the highest quality data at a low cost per sample.

3130 Genetic Analyzer

Applied Biosystems 3130 Genetic Analyzer is the latest generation of 4-capillary electrophoresis instruments for the low to medium throughput laboratories. The system offers industry-leading performance, plus sophisticated automation capabilities allowing you to save time, reduce costs and increase productivity.

3130xl Genetic Analyzer

Applied Biosystems 3130xl Genetic Analyzer is the latest generation of 16-capillary electrophoresis instruments for the medium throughput laboratories.

310 Genetic Analyzer

The ABI PRISM® 310 Genetic Analyzer is an automated single-capillary genetic analyzer designed for a wide range of sequencing and fragment analysis applications.

Step 6: Analyze Data

Analyze data with AFLP-specific analysis module in GeneMapper software. The sizes of the detected restriction fragments are then compared with reference data in t-RFLP databases.

Products for Step 6: Analyze Data

GeneMapper® Software

GeneMapper is a flexible genotyping software package that provides DNA sizing and quality allele calls for all Applied Biosystems electrophoresis-based genotyping systems.
Literature
Application Note
- Terminal Fragment Length Polymorphism (T-RFLP) Analysis on Applied Biosystems Capillary Electrophoresis Systems: T-RFLP on the 3130/3730