Description
The former core facility for DNA sequencing and genotyping at CMM L8:00 and the DNA sequence facility, KISeq, established at CMB 1992, merged year 2008 into the new core facility KIGene, genetic analysis at CMM. We are located at the Department of Molecular Medicine and Surgery, the Neurogenetics Unit, CMM L8:00, Karolinska University Hospital at Solna.
Our vision is to provide a high quality, reliable, high efficiency and flexible core facility, aiming at providing the right solution and study set-up for the right project.
The facility houses several different instruments (see below), and in order to meet the increasing demand on higher throughput and efficiency, as well as on cheaper analyses, our core-facility has introduced several options for gene expression analysis and the analysis of genetic variations including SNPs, microsatellite markers and insertion/deletion polymorphisms.
Most of our equipment can be used by those who have sufficient knowledge and experience. The facility provides start up theoretical and technical assistance. For some applications, we also offer full-service.
We offer:
- Sequencing services with a turn-around time of less than 24 hours
Genotyping Expression analysis Quantity and quality determination of RNA, DNA, protein and dye incorporation
- DNA methylation analysis
Flexible services - three service levels:
Infrastructure/methods
The ABI 3730 and 3130XL PRISM® DNA Analyzers for:
- DNA sequence analysis
- fragment analysis
The ABI 3730 and 3130XL PRISM® DNA Analyzer enables high quality sequencing up to 700 bases and high resolution (1 bp) fragment analysis. The ABI 3730 instrument is in the 96 or 384 well format using a 48 x 36cm capillary array. The ABI3130XL is in the 96 well formats using a 16 x 36cm capillary array.
For DNA sequencing we have the appropriate matrices set up on the instruments for BigDyeTM terminators v.3.1 and 1.1 (Applied Biosystems) and DYEnamicTM ET terminator (Amersham Bioscience). We strongly recommend the use of the BigDyeTM terminator kits.
For fragment analysis the spectral matrixes are based on DS-33 (recommended) or DS-30. Dye set DS-33 allows analysis of fragments labeled with four different fluorescence dyes: 6-FAM, VIC, NED and PET. The fifth dye, LIZ, is reserved for the internal size standard. DS-30 allows analysis of fragments labeled with 6-FAM, HEX and NED using ROX as size standard.
We provide three service levels on the ABI3730 instrument. More information is available in the pdf below.
ABI TaqMan® OpenArray® genotyping system for:
- real-time gene expression analysis
- SNP analysis (end-point analysis)
The TaqMan® OpenArray® system provides PCR-based SNP analysis and gene expression in a medium- to high-throughput format. OpenArray® technology uses proven TaqMan® Assay chemistry and nanoliter fluidics.
TaqMan® OpenArray® plates are delivered from Applied Biosystems containing the selected TaqMan® assays pre-loaded and dried down in the through-holes. The samples are mixed with the TaqMan® OpenArray® master mix and loaded onto the plate. After sealing the plate, it is cycled and, for a genotyping plate, imaged. This simple workflow enables one person to generate more than 50.000 genotypes or 25.000 expression analysis data points in a single day without the use of robotics.
Each TaqMan® OpenArray® plate contains 3,072 through-holes arranged in 48 subarrays of 64 through-holes each. The OpenArray® Autoloader can load one, two or three samples onto each subarray. Six different TaqMan® OpenArray® Genotyping Plate formats enable researcher to test from 144 samples against 16 unique TaqMan® Genotyping assays to 12 samples against 256 assays.
Five available custom OpenArray® Real-Time PCR Plate formats enable to test from 48 samples against 18 unique assays to 12 samples against 224 assays. The platform can be used to run both TaqMan® and SYBR® Green assays.
The core facility will provide start up theoretical and technical assistance for "hands-on" users. Each user reserves a date and time for occupying the instrument by a booking system. More information is available in the pdf below.
The ABI 7900HT instrument
- real-time gene expression analysis
- SNP analysis (end-point analysis)
The ABI 7900HT enables SNP analysis by dissociation analysis or allelic discrimination as well as gene expression analysis using the 5nuclease (TaqMan®) assay. The instrument operates in the 96, fast 96 (PCR in 30 minutes) and 384 well format. The instrument has the equipment to run the micro fluidic card (low-density array).
The core facility will provide start up theoretical and technical assistance for "hands-on" users. Each user reserves a date and time for occupying the instrument by a booking system.
PSQ96MATM and Pyrosequencing Assay Design Software
- up to tri-plex SNP analysis
- microsatellite scoring
- haplotyping
- CpG-methylation analysis
- allelic frequency quantification
- short sequence analysis
PyrosequencingTM is a well established technology for DNA analysis, with emphasis on SNP analysis. The Neurogenetics Unit has evaluated the Pyrosequencing method and the PSQTM 96 instrument for the analysis of genetic variations [Nordfors et al. 2002. Hum Mut 19(4): 395-401], which showed that the instrument is very robust and has a > 99% accuracy. The instrument is user-friendly, robust and fast (96 samples are analysed within 10 min). The CF has now updated the PSQTM96 instrument into a PSQTM96 MA (Multi-application), enabling tri-plex SNP-analysis, haplotyping, microsatellite scoring, CpG-metylation analysis, allelic frequency quantification and short sequence analysis. The new Assay Design Software helps you choose your optimal PCR- and pyrosequencing primers.
The core facility will provide start up theoretical and technical assistance for "hands-on" users. Each user reserves a date and time for occupying the instrument by a booking system.
NanoDropTM ND1000 Spectrophotometer
- small volume quantity and quality determination of DNA, RNA and proteins
Thermo Scientific NanoDropTM 1000 Spectrophotometer enables highly accurate UV/Vis analyses of small volume (1 µl) quantification of DNA, RNA, proteins and dye incorporation. We only offer hands-off service on this instrument.
Nanodrop samples can be placed in the mailboxes at CMB or CMM before 13:00 or brought directly to KIGene. You will receive the stickers by internal mail. Samples will be stored a month in our freezer.
Practical information
Please see our prices and more information in the pdf below. Order forms and more information about DNA sequencing (including troubleshooting): www.ki.se/kiseq/
