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Marine
DNA Sequencing and Analysis Facility The Marine DNA
Sequencing and Analysis Facility was initiated in 1999 with grants from the
Maine Science and Technology Foundation to provide molecular biology core
services to MDIBL investigators as well as to scientists at other non-profit
institutions. An award from the
National Science Foundation in 2001 supported the addition of real-time
quantitative PCR analysis. In 2002,
RNA analysis and DNA microarray printing and
scanning instruments were added, funded by the Expressed Sequence Tag Projects In 2003, three normalized cDNA libraries were produced from multiple tissues of the
dogfish shark Squalus acanthias,
the little skate Leucoraja erinacea, and the lobster Homarus
americanus, as the basis for gene
identification through the generation of expressed sequence tags. In 2004, additional normalized cDNA libraries were produced from multiple tissues of the
green shore crab Carcinus maenas and
the killifish Fundulus heteroclitus,
rectal gland of Squalus acanthias,
liver of Leucoraja erinacea, and
whole copepod Calanus finmarchicus. An automated colony picker was added in
2004 to facilitate processing of these libraries. Sequence data
and blast results for MDIBL’s EST projects are made
available on a timely basis through GenBank (www.ncbi.nlm.nih.gov). Production of ESTs
at MDIBL is summarized here. Interactive blast searches and other computational
analyses of these data are available at http://decypher.mdibl.org. Samples of
library clones or plasmid preparations may be obtained upon request and
completion of a Materials Transfer Agreement.
Please contact dtowle@mdibl.org or cmsmith@mdibl.org. The DNA
Facility maintains an updated protocol manual for basic molecular methods,
available at www.mdibl.org/~dtowle/mbw. Two ABI 3100 sixteen-capillary sequencers offer 24-hour
turn-around for most investigator-submitted samples. Submission information is available online. A BioMek 2000 liquid
handling robot is available for plasmid preparations and other moderately
high throughput procedures. An Agilent 2100 Bioanalyzer is available for analysis of RNA quality and
quantity. A microfluidics
chip and fluorescence monitoring allow electrophoretic
analysis of 12 samples per 30-minute run.
Typical RNA preparations are diluted 10X and 1 μl
of the dilution is used for analysis.
Specific protocol information is available at online.
Single-gene expression analysis is offered by the DNA
Facility using the Stratagene MX4000 and MX3005P
Real-Time Quantitative PCR systems. The
most straightforward method is SYBR Green binding, which allows the use of oligonucleotide primers developed in the investigator’s
laboratory for conventional PCR and does not require the synthesis of a TaqMan probe or molecular beacon. Two commercially available kits have proven
to be robust and quantitatively responsive: Qiagen’s
Quantitect SYBR Green PCR Kit and Stratagene’s SYBR Green QPCR Master Mix. The investigator mixes cDNA
template, primers, and SYBR Green PCR reagents in specially-designed microtube strips. Stratagene 410022 tubes and 410024 caps MUST be used in the MX4000 to
avoid jamming the sample scanning mechanism!
For the MX3005P instrument, DIFFERENT TUBES must be used: Stratagene 401428 tubes and 401425 caps. DNA
Facility staff will assist with setting up the MX4000 or MX3005P instrument
according to investigator specifications. Full service runs are also
available, in which the investigator provides template, primers, and probe
(if necessary) and the facility staff sets up the reactions. Experimental design and data analysis may
be carried out in consultation with staff.
Specific protocol information is available online. The DNA Facility serves as a centralized ordering site for
oligonucleotide synthesis provided by Integrated
DNA Technologies, Inc. On-site
investigators may take advantage of the fee structure established with
IDT. An order form is available online. A Genetix QPix2 robotic colony
picker is available for automated collection of bacterial colonies or phage
plaques from agar plates. It can also
be used to re-array libraries. The
picking capacity is more than 10,000 colonies per day. Consult staff to arrange for training and
use. A GeneMachines OmniGrid Accent arrayer with 32
Parallel Synthesis silica spotting pins is equipped to print up to 20,000 DNA
spots on a treated microscope slide.
Three 384-well plates can be processed simultaneously without manual
intervention, printing up to 50 replicate slides during a run. Investigators supply their library clones,
PCR products, or oligonucleotides in a format
specified by the DNA Facility staff. Oligonucleotide (50-mer) arrays are currently available as follows: Fundulus heteroclitus: 617 features manually
selected by the Fundulus Genomics Consortium from
EST libraries, printed in 8 replicates per slide Homarus americanus: 2,313 features selected
from a normalized EST library, printed in triplicate in 2 arrays per slide Carcinus maenas: 4,462 features
representing contigs and singletons from a
normalized EST library, printed in duplicate per slide For hybridization analysis, investigators may supply total
RNA from their tissues of choice or may provide snap-frozen or RNAlater-preserved tissues for preparation of RNA
by the DNA Facility staff. Labeling of
cDNA probes and hybridization to microarray slides may be performed by investigators or by
the staff, as desired. A BioMicro 4-bay Binding of labeled cDNA to the
printed arrays is visualized on an Axon GenePix
4000B scanner and the resulting images are analyzed with GenePix
Pro and Acuity software in consultation with the DNA Facility staff. A brief protocol of proven microarray
techniques is available online.
Contacts David W. Towle, Ph.D., Director
of the Marine DNA Sequencing and Analysis Facility Phone
207-288-9880 x474 Email
dtowle@mdibl.org Christine M. Smith, B.A., Supervisor of the Phone
207-288-9880 x130 Email
cmsmith@mdibl.org Updated
Supported by National Science Foundation Atlantic Canada Opportunities Agency
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