Molecular & Cell Biology
The Molecular and Cellular Biology Core (MCB Core) provides services, reagents and training for many aspects of molecular biology, cellular biology, and cell culture. Molecular biology services include both capillary (ABI 3730xl) and Next-Gen (Illumina MiSeq) sequencing, high throughput genotyping, robotic preparation of RNA and DNA, preparation of monkey genomic DNA, 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, real-time PCR machines, scanning fluorometers and phosphor imaging. Cell biology 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. the core also produces lentiviral vectors for gene expression, shRNA expression and fluorescent reporter gene expression.
Next-Generation Sequencing (Illumina MiSeq)
The MCB Core offers next-generation sequencing for smaller projects that do not require the throughput of the HiSeq 2000 that is offered by the OHSU Integrated Genomic Laboratory.
Frequently asked questions
What kind of sequencing run MiSeq can do?
Do you provide library construction service?
Typically we do not. We will however offer advice and guidance and point you to services that will create libraries.
MiSeq sample drop-off
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
Do you provide library quantification service?
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.
What is the MiSeq sequencing turn time?
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 DropBox or even provided to you on a USB data stick.
Capillary Sequencing (ABI 3730xl)
Capillary sequencing remains the method of choice for small scale sequencing projects and construct validation. 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.
- 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.
Small and Medium Scale Robotic Nucleic Acid Preps (RNA, genomic DNA, minipreps)The Molecular Cores 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. We anticipate RNeasy, QIAquick and AllPrep kits will be 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.
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.
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 LifeTechnologies 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 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 providign 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 Water (Milli-Q Advantage)
- Technical support and training.
- 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
MCB Internal Rates (OHSU login required, click on Molecular and Cell Biology Rate Sheet)