The Molecular Technologies Support Core (MTC) provides technology-driven services in support of molecular biology and cell biology for ONPRC investigators. The goal of the MTC is to provide flexible, state-of-the-art, time and cost-effective technologies to assist molecular and cellular biology through a carefully chosen balance of both core-delivered services and core-supervised, customer self-service.
DNA and RNA-based services include NextGen and capillary DNA sequencing, automated genotyping, robotic preparation of RNA and DNA, preparation of monkey genomic DNA, generation of lentiviral vectors, generation of monkey specific cDNAs, establishment of monkey-specific real-time PCR assays, large-scale plasmid and phage preparation, supply of a limited number of reagents, training and access to specialized equipment including liquid handling robots, automated nucleic acid preparation, real-time PCR machines, scanning fluorometers, plate readers and a variety of nucleic acid quantification/quality assessment platforms.
Cell-based services include preparation of media and reagents, freezing, storage and amplification of cell lines, transfection and cloning of cell lines, establishment of primary cell lines, preparation of coated culture ware and cover slips, and training of new personnel.
Next-Generation Sequencing (Illumina MiSeq)
The MiSeq is very flexible, running both v2 and v3 chemistries and newly introduced are economically prices micro and nano kits for very small scale projects.
|Read Length||1 x 36 bp||2 x 25 bp||2 x 150 bp||2 x 250 bp|
|Total Time||~4 hrs||~5 hrs||~24 hrs||~39 hrs|
|Output||540-610 Mb||750-850 Mb||4.5-5.1 Gb||7.5-8.5 Gb|
|Read Length||2 x 75 bp||2 x 300 bp|
|Total Time||~21 hrs||~56 hrs|
|Output||3.3-3.8 Gb||13.2-15 Gb|
|Nano and micro kits|
|v2 Micro||V2 Nano|
|Read Length||2 x 150 bp||2 x 150 bp||2 x 250 bp|
|Total Time||~19 hrs||~17 hrs||~28 hrs|
|Output||1.2 Gb||300 Mb||500 Mb|
Typically we do not, but will upon request. We will however offer advice and guidance and point you to services that will create libraries.
Provide at least 20 µl of 2 nM library DNA for MiSeq sequencing. Main campus labs can send cold packages through campus mail to: MCB Core, Cooley Building, ONPRC, Mail code: L584
Illumina and the core strongly recommend that all libraries be quantified prior to MiSeq runs. Libraries can be quantified by qPCR (best) or by Qbit fluorometric analysis. Fragment size can be analyzed on a high sensitivity Bioanalyzer DNA chip. The core offers all these services.
Turn-around time varies depending on the kind of sequencing kit used. Usually it is 2-3 days after we receive samples.
The core recommends that users establish a free Illumina BaseSpace account. Data from MiSeq runs can then be uploaded directly to your account. Alternately data can be deposited on an intranet shared drive, uploaded to Box, or even provided to you on a USB data stick.
The MTC does not, but we work very closely with the ONPRC Biostatistics and Bioinformatics Core (BBC) to ensure that users get the bioinformatics support they need. We also encourage our users to consult with the BBC prior to starting projects to ensure appropriate sample number and analytic approaches are used.
Capillary sequencing (ABI 3730xl)
The nature of NHP research often creates the need for small scale sequencing projects. Capillary sequencing remains the method of choice for small scale sequencing projects and construct validation. The MTC core is now one of only two providers of capillary sequencing at OHSU. Average run length is about 800bp.
- DNA sequencing is performed on a 96-capillary ABI3700XL. Combined with a robotic pipetting station, this allows the core to offer extremely high quality, low cost sequencing.
- Well established protocols for amplicon sequencing and targeted resequencing and mutation detection.
- Special pricing available for high volume or user reaction setup.
- We gladly assist in helping with DNA preparation protocols and data analysis.
- We specialize in working with scientists to optimize protocols for hard to sequence regions.
- Quality and satisfaction guaranteed and DNA will be re-sequenced at no cost if quality is not acceptable.
Single cell sequencing
The core maintains a DropSeq sytem for single cell sequencing. Users provide the isolated cell and the MTC does the encapsulation in oil droplets and assists with library preparation as needed. The core can also assist with cell isolation and solubilization if desired. Depending on library size users are directed to the appropriate sequencing platform. The MTC also works closely with the ONPRC Biostatastics and Bioinformatics Core to ensure that customers are directed to appropriate bioinformatic resources.
Small and medium scale Robotic Nucleic Acid preps (RNA, genomic DNA, minipreps)
The Core's QIACube and Maxwell 16 MDx instruments provide medium throughput, automated nucleic acid extraction and/or clean-up. The systems automate a large variety of existing sample kits at prices comparable to manual preps. The QIACube automates most of the Qiagen MINI scale kits. The RNeasy, QIAquick and AllPrep kits are most popular. The Maxwell is great for larger genomic DNA preps up to 1 ml of buffy coat in less than 1 hour. Contact CoreyAyne Singleton for additional information about your purifications of choice and training. Users are encouraged to be trained and perform analyses themselves.Fully automated DNA, RNA, and minipreps can also be performed on the cores Eppendorf EpMotion 5075 Vac system. This system provides true, walk-away nucleic acid preps in 8-well or 96-well formats. The system can also be used for robotic preps of other assays that are performed in high volumes. The core will train users in the use of the system if desired.
Nucleic acid quantification and quality control
Garbage in –Garbage out. Careful quantification and quality analysis is essential for RNA and DNA preparations prior to all molecular procedures, especially so for library construction, chip analyses, sequencing projects and RNA analyses. To help ensure investigators achieve needed quality standards, the core provides a nanodrop for simple spectrophotometric analysis, 2 Qubits for rapid fluorescent quantification, fluorescent plate reader for larger numbers of quantification, and an Agilent Bioanalyzer for analysis of RNA and DNA purity and size. For all these instruments, training is provided and customers are encourage to do it themselves.
Quantitative PCR, genotyping and QuantStudio Open Array
The core provides equipment and technical assistance for performance of quantitative PCR (real-time PCR). Equipment supported by the core includes a ABI QuantStudio 12K Flex with 96-well, 384-well, and Open Array options and an ABI 7900HT with 96-well and 384-well plate options. The systems are available for standard qPCR and genotyping (SNP and copy number) and the Open Array option allows high throughput RNA measurement and genotyping for both standard arrays and custom arrays. Examples of standard arrays include microRNA panels, human kinome and cancer RNA panels and drug genotyping panels. The core will train new users on system use and assay design.
Lentiviral vector preparation
The core provides production of 2nd and 3rd generation lentiviral vector production providing either conditioned medium or concentrated virus preparations. The core will assist with vector design as needed and provides some standard vectors. The core can assist with infection of cell lines and cell sorting if desired.
What steps have to be taken before a lentiviral prep can be started?
The core has a very specific checklist of requirements that must be met before actual production of lentivirus can begin:
- The lentiviral core must receive a copy of your current RDRQ with proposed constructs and an electronic (.pdf) copy of IBC approval letter before any work can be started.
- Generation of plasmid for lentiviral preps should be transformed in XL-010 gold, STBL 3 or comparable competent cells. Plasmid must be endotoxin free. (We get good results with Qaigen Maxi kits.) This step can also be done by the Core facility if you wish.
- Quality control digestion of plasmid should be done to confirm correct band cuts/pattern expected. For any labs doing steps 2 and 3 themselves, they will need to send a copy of gel image to the core for records.
- We strongly suggest doing a test plate of all plasmids to confirm through FACS or qPCR of successful packaging of viral vectors before doing a full scale prep. This will help prevent doing a large scale prep that may not package well. Once the Core starts a full scale prep all charges will be incurred.
- Actual production of lentivirus can begin at selected scale (see below, 10 µg of DNA is needed for each 10cm dish reaction.)
- The Core will gladly work with you to set up any custom preparations that you have in mind.
Other cell biology services
- Semi-automated large-scale genomic DNA preparations.
- Freezing, thawing, and amplification of cell lines.
- Preparation of custom cell culture medium.
- Provision of specialized supplies, cell culture ware (plain and coated) and reagents.
- Assistance with special projects.
- Ultrapure DNase, RNase-free Water (Millipore IQ-7000)
- Technical support and training.
- Robotics (Eppendorf EpMotion 7075)
- DNA purification: QiaCube, Maxwell 16 MDx
- qPCR sytems
- Calcium kinetics and other time-based fluorescent assays using a Molecular Devices FlexStation
- Molecular Devices M5 multifunction plate reader (absorbance, fluorescence, luminescence)
- Gel Doc System
- Nanodrop spectrophotometer
- Countess Cell Counter
- QBit fluorometer
- Bioanalyzer for RNA and DNA quality
- Shaking Incubators
- Technical support and training provided for use of all instrumentation