Plant-Microbe Genomics Facility

The Facility can perform Genotyping by fragment analysis. The Facility has a 3730 DNA Analyzer (Applied Biosystems, Inc.) that can analyze DNA fragments labeled with a fluorescent dye to determine the size of the fragment with a resolution of one base. With 48 capillaries the instrument can analyze up to 48 samples simultaneously as well as accommodate 96-, or 384-well plates. The 3730 DNA Analyzer has the ability to multiplex within one capillary by the use of 5 different fluorescent dyes, and/or differing fragment size.

The Facility can perform such techniques as allelic determination, amplified length polymorphism analysis (AFLP), microsatellite analysis (STR and SSR), and single nucleotide polymorphism analysis (SNapshot).

The facility has a CFX96 Real-Time Detection System (BioRad) which can accurately quantitate PCR products in real time. The CFX96 system is very sensitive and can simultaneously measure up to 6 different PCR products in each of 96-wells. This system is adept at precisely measuring the concentration of specific RNAs, measuring gene copy number as well as perfoming other applicatons that require the measurement of specific DNA molecules. The CFX96 system can analyze all of the current detection chemistries, such as SYBR Green, Taqman, etc. In addition, the instrument can be used as a specialized fluorometric based plate reader that can detect most dyes for applications such as high resolution melt analysis, single nucleotide polymorphism detection, and measuring protein melting.

The service includes the use of the CFX96 system and a file with the results. In addition, the Facility can provide a copy of the CFX96 software at no cost to you. The Facility will provide the 96-well plate and optical film which is included in the per run cost. The customer will be responsible for providing all other consumables (e.g. primers, polymerase, nucleotides, etc.) and setting up the reactions in the plate.

Sanger Capillary
The facility uses the 3730 DNA Analyzer from Applied Biosystems, Inc. and BigDye® Terminator Cycle Sequencing chemistry. This automated instrument utilizes fluorescent dye labeled nucleotides and provides high volume capabilities, e.g. 384 sequencing reactions in 12 hours, and typically yields 800 to 1000 bases per reaction depending upon the quality and nature of the template

The Facility has a 3730 DNA Analyzer (Applied Biosystems, Inc.) that can analyze DNA fragments labeled with a fluorescent dye to determine the size of the fragment with a resolution of one base. With 48 capillaries the instrument can analyze up to 48 samples simultaneously as well as accommodate 96-, or 384-well plates. The 3730 DNA Analyzer has the ability to multiplex within one capillary by the use of 5 different fluorescent dyes, and/or differing fragment size.

The Facility can perform such techniques as allelic determination, amplified length polymorphism analysis (AFLP), microsatellite analysis (STR and SSR), and single nucleotide polymorphism analysis (SNapshot).

The facility uses the 3730 DNA Analyzer from Applied Biosystems, Inc. and BigDye® Terminator Cycle Sequencing chemistry. This automated instrument utilizes fluorescent dye labeled nucleotides and provides high volume capabilities, e.g. 384 sequencing reactions in 12 hours, and typically yields 800 to 1000 bases per reaction depending upon the quality and nature of the template.

The Facility has a GE Healthcare Biacore T100 that can measure the interaction between proteins and proteins, DNA and proteins, metabolites and proteins, as well as other biomolecules that can be anchored to a gold covered chip.The measurements are done in real-time with no labels and utilize surface plasmon resonance (SPR) technology. The T100 can measure concentration, binding specificity, and affinity constants.

Due to the nature of the studies performed with the T100, the client uses the instrument in order to allow for instant analysis of the results and therefore immediate decisions about the next step or experiment to be done. In order to use the instrument, all clients are required to attend a training session provided by the Facility or the Biacore Basics course offered by GE Healthcare. The training provided by the staff is done at the Facility, takes one to three days, and can accommodate up to 2 clients at a time. The client will need to provide all materials and training will utilize the client's samples. An experienced staff member will provide guidance during the initial experiment and the training has a set fee. The training will include: practical use of the instrument, experimental design, and analysis.

The Facility is pleased to announce a new service - Selective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE). SHAPE is a method of RNA analysis that is used to determine RNA structure at the nucleotide level. RNA molecules are combined with a hydrolyzing agent, such as benzoyl cyanide (BzCN), which selectively hydrolyzes unpaired nucleotides, forming 2’-O-adducts. RNA molecules then undergo a primer extension reaction, which the adducts inhibit, forming cDNA fragments that terminate at the location of hydrolysis. These fragments are analyzed on the 3730 DNA Analyzer (Applied Biosystems, Inc.) along with a negative control and one or two dideoxy sequencing reactions. The electropherograms from these reactions are aligned with each other, as well as a reference sequence, to determine the identity of unpaired nucleotides in the original RNA molecule.