|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
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! 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 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
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
