GROUP
ONE AND TWO RESEARCH JOURNAL
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July 20 |
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July 27 |
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July 30 |
July 31 |
Tuesday - July 15, 2003 - Introduction to Group Research
Groups one and two are working collaboratively on the phylogenic relationship of certain metazoans collected on the New Jersey shore at Island State Park. Our mentor Dr. Jim Bonacum gave us an overview of evolutionary biology and reviewed several models used in phylogenetic research (i.e. distance methods and statistical methods). Jim also discussed cladistics and presented the advantages and disadvantages of parsimony data. We ended our discussion with the speciation and geographical distribution of Drosophila in the Hawaiian Islands.
Wednesday - July 16, 2003 - Collecting at Island Beach
State Park - Jersey Shore
The entire Woodrow Wilson Biology group journeyed to Island Beach
State Park on the Jersey shore for a day of collecting specimens from
which DNA would be extracted and processed for sequencing. Groups 1 and
2 collected over 60 marine and terrestrial specimens in three different
locations: bayside, ocean-side, and tide pond.
 Silversides collected at the bay |
 Woodies preparing to collect specimens at Barnagat Bay |
 Nature never fails to awe Jim Bonacum |
July
17, 2003 – Identifying, Splicing, Dicing, and Extracting
During our daily morning meeting with Jim, the group decided to use a
division of labor approach using members to key out organisms, label,
photograph, dissect, and extract DNA from our specimens. Established protocols
with modifications were followed for DNA extractions.
 Peggy Alexander and Deborah Moffitt work with samples |
 Docia Generette and Lynda Smith dissect specimens |
 A mole crab specimen |
Friday - July 18, 2003 – All day and all night!!!!!
Most of Friday morning and afternoon was devoted to extracting DNA from
the remaining specimens using the DNAEasy kit from Qiagen. We then amplified
the region of the 18S rDNA gene via PCR. All sixty-three PCR products
we cleaned and purified using the GeneClean Kit from Bio 101.
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 The group poses for a picture well past midnight. |
July
19, 2003 - The first sequence run!
We prepared all gene cleaned products for the sequencing reactions. Thirty-two
of the prepared specimens along with positive and negative controls were
sequenced using a 64 lane polyacrylamide gel and loaded into the sequencer.
Dr. Bonacum analyzed and saved the sequence data for each lane to computer
files on Sunday.
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 64 gene-cleaned specimens |
July 21, 2003 – Oh where, oh where is the DNA?
The group met with Dr. Bonacum for an update on the first sequencing run.
Jim related that the data was mixed. Some lanes contain usable data and
others were either very faint are not readable. Three possible causes
were identified: the templates were not dried properly and contained too
much isopropanol, there was not sufficient DNA template, and perhaps the
sequencer was not reading properly and needed to be recalibrated.
In order to address the problems identified above, a second sequencing
gel was prepared using several modifications. The volume of DNA template
was increased from 1.0ul to 2ul. Six bacterial plasmids from Dr. Lemke’s
group were selected to provide data for his group along with a check on
the calibration validity of the sequencer. Three samples from Dr. Eagan’s
group were included along with 12 DNA templates containing samples from
the first run and selected specimens not yet sequenced.
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July 22, 2003 - Preparing for the second sequencing gel run
Dr. Bonacum met with the group to discuss and demonstrate Sequencher.
Sequencher is a DNA sequencing and analysis software package developed
by Gene Codes Corporation. The package is known for its lightning fast
contig assembly which should greatly facilitate data analysis. Jim has
obtained a site license for Sequencher which will be available on Wednesday,
July 23.
In the lab, certain members of the group finished the final preping of the specimens for the second sequencing gel.
 "Dr. Tormentor" updates the group. |
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July 23, 2003
Certain members of the group loaded Sequencher on their computers and
begin editing viable sequencing data from Gel 1. The second gel was prepared
and loaded with selected DNA templates prepared the day before. Other
members of the group gene cleaned the last 32 of the orginal 64 specimens
to prepare for sequencing gel 3.
 Editing sequenced data using Sequencher |
 Phi Le and Larry Little consult with Justine Cooper. |
 The thermocycler used to facilitate PCR reactions. |
July
24, 2003 -
Most of the morning was devoted to editing data from gels one and two
as well as working on the group website, posterboard and journal. The
entire Woodrow Wilson group attended a symposium at Princeton in the afternoon.
After the symposium, selected members of the group completed the final
sequencing prep to run gel three. The gel was prepared and loaded around
2:00 a.m. Friday morning.
 The esteemed speakers of the Princeton Symposium |
 Escaping and relaxing at Princeton |
July 25, 2003 -
Worked continued on editing and blasting selected viable DNA sequences from gels one and two. At present we have confirmed matches for 6 specimens.
 Edit, align, and BLAST! |
July 26 - Dr. Rob DeSalle gives the lowdown on Sequencher and ClustalX
Dr. Rob DaSalle gave a presentation on the use of Sequencher and ClustalX. ClustalX provides a new window-based user interface to the ClustalW multiple alignment program. It uses the Vibrant multi-platform user interface development library, developed by the National Center for Biotechnology. Alignments can be saved in several formats to generate phylogenetic trees and cladograms.
 Dr. Rob DeSalle introduces the group to ClustalX. |
July 28 - FASTA and Alignment and Panic!
The deadline looms over us all! Certain members are frantically creating FASTA files and carrying out alignments to place into PAUP to generate cladistic analysis. Other members of the group are putting the final touches on the research project, posterboard, and research journal.
 ClustalX screenshot |
 The Woodies in the t-shirts at lunch. |
July 29 - Finally a tree!
Dr. Bonacum sat at his desk in the meeting room, analyzing the data to produce our first tree. There were some problems with it. Some relationships that it produced were not expected. Jim continued working with the data and produced some trees that we could work with.
The Tree
