RGCB has a centralized DNA sequencing facility to meet the demand of scientists in various research fields. This facility is greatly helping their research work, catering to the sequencing needs of the Institute. Automated DNA Sequencing Facility was started with the installation of ABI 310 Genetic Analyzer (a single capillary machine, from ABI) on March 1999. This machine till August 2006 was largely used for sequencing of DNA samples and Gene Scan Analysis. This facility received and installed a new ABI 3730 - 48 capillary high throughput DNA Sequencer (3730 DNA analyser) in June 2006. The fluorescence-based DNA sequencing system, with capillary electrophoresis is having 48 capillaries operating in parallel. The system automatically performs sample injection, gel matrix replacement, DNA separation, detection, and data analysis. This system sequence 48 samples in 2 hours' time and perform up to 12 runs per day. Average read length with big dye terminator chemistry is 800bp. This system can also be used for genotyping and SNP analysis.
The Core accepts DNA samples (pure plasmids, mini-prep plasmids, M13 clones, cosmids, lambda clones, PCR products, gel isolated fragments, bacterial genomic), sequenced PCR products and processes them according to protocols for Applied Biosystems DNA Sequencers (BDT chemestry). One sequencing run (one template, one primer) typically produces 700-900 nucleotides of high-quality sequence data or more, for good-quality template and a well-designed primer. The results are returned in three forms: a raw sequence file, with blue score and a chromatogram file. The sequence file (a flat ASCII text file) is returned to the researcher by electronic mail. All samples and primers must be dissolved in distilled water, and must be at the standard concentrations listed below. We cannot do custom dilutions or reconstitute lyophilized samples
Each sequencing plate includes at least one lane of standard DNA known to generate good sequence. The sample handling and data analysis are extremely reliable and the Core personnel have a very low rate of error. If any problems are indicated in our sample processing, we repeat the affected samples on request. We typically process samples within two days after receipt. Samples are processed Monday through Friday for overnight instrument runs. The data are processed and returned by email to the users.
The Genomics Facility is one among the five highly specialized laboratories located within a class 10 facility at campus 3 (Bio-Nest), providing investigators (and external clients) access to State-of-the-Art genomics capabilities. The genomic facility operates as a core facility offering molecular biology technology support services and training in DNA sequencing, genotyping and gene expression analyses to academic, government and private sector institutions. Services include, DNA Sequencing, Fragment Analysis, Gene Expression Service and DNA bar-coding.The facility has:
The 8-capillary 3500 DNA Analyzer is used for traditional DNA sequencing and DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection. It provides the highest quality data, additionally the machine is In Vitro Diagnostics approved.
The CFX Connect real-time PCR detection system offers two-target analysis and excellent thermal cycler specifications. The system incorporates innovative optical technologies with powerful software to provide maximal reliability and efficiency for every real-time PCR needs.
Dr. Manoj P
Manager (technical services-DNA sequencing)