OHSU

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.


Contacts

Director: Eliot Spindel 503-690-5512
Director of Lentiviral Services: Greg Dissen 503-690-5382
Molecular Biology Lead: Yibing Jia 503-690-5525
Cell Biology Lead: CoreyAyne Singleton 503-690-5568

 

Next-Generation Sequencing (Illumina MiSeq)MCB 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. 

MiSeq Approximate Run Duration and Output

Cluster Generation and Sequencing
Read Length (bp)
Total Time*
Output
1 x 36
~4 hrs
540-610 Mb
2 x 25
~5.5 HRS
750-850 Mb
2 x 100
~16.5 hrs
3.0-3.4 Gb
2 x 150
~24 hrs
4.5-5.1 Gb
2 x 250
~39 hrs
7.5-8.5

Summary of MiSeq rates

Description Cost
MiSeq 50 cycle reagent kit
$798.00
MiSeq 300 cycle reagent kit
$1045.00
MiSeq 500 cycle reagent kit
$1160.00
MiSeq PhiX control (µl)
$15.00
MiSeq run setup and clean up
$50.00
MiSeq data analysis
$60.00 per hour
MiSeq library construction
Inquire
MiSeq Library first quantification
$50.00
MiSeq Library additional quants
$25.00



Frequently asked questions

What kind of sequencing run MiSeq can do?

There are three MiSeq kits to choose from: 50 cycle, 300 cycle, and 500 cycle kits.  All kits can be used to do single read or paired-end (PE) sequencing of libraries.
Run Type
Runtime Bases/read Max number of reads
Total # of bases
SE50 5 h
50 15-17 million
750-850 Mb
PE150 30 h
150 + 150
30-40 million
4.5-5.1 Gb
PE250 50 h
250 + 250
30-40 million
7.5-8.5 Gb

 

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.

Data Transfer

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.
  • Standard pricing:
    • Sequencing (<96 rxns) $4.50
    • Sequencing (97-999 rxns) $4.00
    • Sequencing (>999 rxns) $3.00
    • Sequencing, with better buffer (< 96 rxns) $6.00
    • Sequencing, with better buffer (97-999 rxns) $5.50
    • Sequencing, with better buffer (>999 rxns) $4.00
  • 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.

Robotic Nucleic Acid Preps (RNA, genomic DNA, minipreps)

Fully automated DNA, RNA, and minipreps are 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. 

Description Details Cost
genomic DNA from Tissue (excellent for cultured cells, buccal swabs, mouse tail or ear punches)
Process up to 20 mg tissue samples, yield up to 40 µg (average 20 µg), in format of 8-strips
$35.00 per strip of 8
Plasmid DNA miniprep
8-well strip or 96-well format, 1.5 ml bacteria culture yields approximately 5-15 µg/well
$29.00 per strip of 8 or $250.00 per 96-well plate
Genomic DNA from Blood
200 µl of whole blood, average yield 5 µg/sample, in format of 8-strips
$38.00 per strip of 8
Total RNA from Blood
300 µl of whole blood, average yield 3-7 µg, in format of 8-strips
$50.00 per strip of 8
Total RNA from tissue or cell culture
10-30 mg of tissue or up to 2 million cells, can yield up to 40 µg of total RNA, in format of 8 strips
$50.00 per strip of 8

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.
Description Cost
1 ml "in stock" lentivirus conditioned medium
$7.50
10 µl "in stock" lentivirus
$60.00
Lentiviral custom preparation
$50.00
Lentiviral Prep concentrated, 4 plates
$300.00
Lentiviral Prep concentrated, 12 plates
$600.00
Lentiviral Prep concentrated, 24 plates
$1000.00
Lentiviral Prep, conditioned medium, 1 plate (10 ml)
$50.00
Lentiviral Prep, conditioned medium, 4 plates (40 ml)
$200.00
Lentiviral Prep, conditioned medium, 12 plates (120 ml)
$350.00
Lentiviral Prep, conditioned medium, 24 plates (240 ml)
$600.00
Lentiviral Prep, Invitrogen "Fast'Titer" reagent surcharge
$40.00

All Rates and Supply costs: 

MCB core rates

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.

Instrumentation

  • 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