Egan’s Einsteins
Research Journal DAY 1: Meeting with
Research Team Dr. Mary Egan, specialist in ancient DNA and classification
of organisms, introduced herself to the team.
Dr. Egan presented her recent findings on tracing haplotypes
of the Muntjac, Right Whales and Bowhead Whales to their common ancestors. Members of our team brainstormed in the fields
of Genomics, Evolution and Phylogenetics
for possible research topics. The team will finalized their exact research
focus after the collection of specific marine organisms from Trip to This State Park is divided into 3 areas -- northern, central
and southern with almost nine-and-a-half-miles of beach front to enjoy.
The
southern natural area, primarily a wildlife sanctuary, extends
almost five miles south to the Barnegat Inlet. We enter the park at
the northern side of the Source: http://www.beachcomber.com/Njshore/Ocean/Islbe/Graphics/isbea.html Some members of the team entered the ocean to collect samples while remaining members were responsible for numbering, cataloguing and temporary storage of samples. We were amazed at the abundant number of diverse organisms collected. The table below lists the 23 specimens and their location.
Day
3: In the morning, the group sorted and preserved the samples by adding 95% Ethanol (EtOH) or by freezing to preserve the DNA for future extractions.. We designed a labeling system for the samples and used an Excel spreadsheet for are recording. Below is the outline of the protocol used for specimen sorting and identification.
5. The DNA specimens were refrigerated for future
analysis. DNA was extracted from all collected samples, following the protocol used for the DNase Easy Kit. Below are photographs of the materials used for the DNA extraction?
Day 4: July 18, 2003
Figure 1 I---------------amplified with S2 & 9R------------I 18S1F S2-> X-----------------------------------------------------------------------------------X <- 5R <- 18S9R I---amplified with 1F & 5R--I I-----------------------amplified with 1F & 9R--------------------------------I Day 5: The best amplified PCR samples were chosen based upon a single band on the agarose gel. Each selected sample was “gene cleaned”. Gene cleaning is nessasary to remove all PCR reagents from the amplified DNA samples. This step is necessary before running a sequencing reaction on the DNA specimens. Day 6: DNA samples were amplified by PCR. A new aliquot of the reverse primer 18S9R was used instead of the old aliquot of the same reverse primer. The conditions for the PCR reactions were the same except for amount of template added. PCR amplified products that showed a faint band on agarose gels received 5 µl of template. The following spreadsheet outlines the exact contents of each PCR reaction sample. Day 7:
Day 8: The group analyzed the bands on the gels with imput from Mary. The best samples were chosen for DNA sequencing. The following spreadsheet was used for the sequencing reactions Day 9: The group gene cleaned the samples for DNA sequencing and set up the sequencing reactions (see protocol for sequencing reaction). Mary gave a tutorial on the use of the software program called Sequencher. This program allows for easy editing and alignment of DNA sequences. Day 10: The sequencing samples were prepared for loading on
a sequencing gel. The following protocol was used to prepare the samples. In the afternoon the Woodrow
Wilson Biology Institute participants traveled to Day 11: The team had good PCR products and yet the sequencing
gel did not show any DNA. We
discussed with our mentor all of the possible factors that could have
brought this about. We brainstormed regarding skills, extraction
methods, and gene cleaning etc.
our mentors Mary and Rob told us that they needed time to troubleshoot”,
to do their own investigating, and to bring their minds together. In trouble shooting NaI became
a target. All reagents were checked. All
PCR machines and primers
were checked for accuracy. Because we got good PCR products, we can assume
that something was wrong with the reagents used in gene cleaning precipitation.
We had a problem therefore we
used a different stock of NaI.(Saturday and
Sunday became a day of reflecting, a day to see family and friends,
a day for revisiting Day 12, Yes, our results were perfect. The e- gels were beautiful!
Beautiful PCR products! YES!
Yes! What a beautiful feeling! , The team met for
discussion to plan our next phase of analysis.
Dr. Egan gave a hands-on lecture to the team using the software
“Sequencher”. Team members utilized the demo program of Sequencher to become familiar with the program. Day 12: Today the team gathered in
our meeting room to analyze our data and gels. The team compared the
aligned sequences to create a phylogenic tree.
The team assembled a matrix and aligned the sequence in the matrix
for phylogenic analysis and produced the phylogenic tree. The rest of
the day was spent finishing and polishing the website, the poster, the
lesson study, and the research presentation.
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