Technical Log
Name: HaoQi (Esther) Li
Date: 6/26/06 Monday
Work Location: Naval Medical Research Center, Spring Field, MD
Title of Project: Developing an Optimized qPCR for Borrelia lonestari
1. Objective – Why did you hope to accomplish today?
I hoped to carry out the PCR process by myself successfully and start working with
qPCR.
2. Events – a. summarize the schedule of events. Procedures performed in detail,
results, analysis, with tables, charts, diagrams, and scanned sketches
b. summarize communications, emails (attached), conversations, pertinent material
read (annotated bibliography)+ reactions, browser used (search identifier,
annotation)
In the morning, I organized my notebook. When Dr. Ju came in, she told me to print out
the ClustalW Borrliea sequences alignments.
Then I did another PCR practice with procedures exactly identical to the PCR practice
from 6/22 and 6/23 except for the concentrations and PCR temperatures. The date is 6/23
Date 6/23/2006
Final Conditions
Vol. Template
Vol. Reaction
Forward Primer
Reverse Primer
dNTP
MgCl2
Reagent
Water
10X Buffer
dNTP
O.tsu630F
O.tsu.747R
Gold Taq
Specificity: O.tsu.r47 gene
PCR
Stage 1: Hold 95C: 10 min.
1l
Stage 2: 3-Temp cycle repeat for 45 minutes
25l
94C: 30sec.
0.3M
58C: 30sec.
0.3M
0.2mM
72C: 30sec.
1.5mM
Stage 3: Hold 72C: 7min.
Stock
10X
2mM
10M
10M
0.3l/each
Total (Before Template Addition)
Each (Before Template Addition)
because that's when the paper was printed.
No. Samples: 4
Master Mix
68.8l
10l
10l
3l
3l
1.2l
96l
24l
There were three lanes of plasmid Ktr47 DNA again: the negative control with no DNA,
the 103copies/l in Tube A, and the 105copies/l in Tube B, exactly like last time.
When the DNA samples were being amplified in the PCR machine, all of the Rickettsia
Department’s SEAP students went to meet Dr. Richards, the department chair, who just
returned from abroad. Dr. Richards helped us to understand our projects by answering
questions. For my project, he explained that Borrelia is not Rickettsia and they are not
related at all other than that ticks transmit them both. Thus I’m working in the Rickettsia
Department but on a project that deals with Lyme disease!!!
http://en.wikipedia.org/wiki/Lyme_disease and http://www.aldf.com/lyme.shtml have a
nice overview of Lyme disease.
Here is a picture of the lone star tick, which can carry the spirochete (spiral shaped)
bacteria Borrelia lonestari.
http://www.aldf.com/images/img-Lone-star-tick.jpg
[///Things I learned for Rebecca’s project.
Dr. Richards explained how ELISA (enzyme-linked immunosorbent assay) worked,
which can be found http://en.wikipedia.org/wiki/ELISA. (I <3 Wikipeida!!!)
For Rebecca’s specific project, the Rickettsia typhi is going to be put in and attached to
the special protein attractive tube. Then in turn its antibody is added so, and finally an
anti-human-antibody (i.e. produced in another animal) is added. When the enzyme
ABTS 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and hydrogen peroxide
H2O2 are added to this long chain of proteins and antibodies, the enzyme ABTS breaks
apart and becomes green.
Sometimes when a disease produces a lot of antibodies, the ELISA can be used but
instead of first adding a protein (IgM, IgG, IgA, IgD, or IgE), the antibody is directly
added on and a protein can be attached secondly. However for Rickettsia, most of the
bacteria migrates through the endothelial (blood vessel) cells so not a significant amount
is found in the blood stream.
///]
After a short lunch, we finished up the second PCR practice. Unfortunately, the DNA
ran off the gel. 
I observed the mentor’s aid Joey doing a real time PCR, i.e. qPCR. Everything is the
same as the PCR, except for the concentration number, some reagent, and the machine
and tubes used to amplify the PCR.
Date 6/26/2006
Final Conditions
Vol. Template
Vol. Reaction
Forward Primer
Reverse Primer
Probe
dNTP
MgCl2
Platinum Taq
Reagent
SuperMix-UDG
Water
O.tsu630F
O.tsu.747R
FAM Probe
MgCl2
Specificity: O.tsu
1l
25l
0.1M
0.1M
0.2M
0.2mM
5mM
0.75 U
Stock
2X(w/ Mg 3mM)
10M
10M
10M
50M
Total (Before Template Addition)
Each (Before Template Addition)
No. Samples: 4
Master Mix
50l
38l
1l
1l
2l
4l
96l
24l
Then just like the regular PCR, three 24l are taken out and put in special qPCR tubes
with lenses at the bottom. After the addition of 1l of either water or O.tsu DNA, the
three tubes are placed in the special qPCR machine and the qPCR program is turned on.
After setting the program to read the specific three tubes and entering in the information
about the three tubes, the qPCR process was started and the lines began to show after a
few minutes.
At first, all three tubes have values above the "threshold" (which means "positive"), but
after 15 minutes the machine adjusted the values and all three lines fell to 0 as normal for
before having been massively multiplied.
We will take the results tomorrow.
---A flood warning was distributed and 3" of rain was expected by 5pm (didn't happen), so
the commanders gave out the "59 minutes" warning so that people can leave early. ("59
minutes" because it is less than an hour, which is okay for a work day).
3. Reflections
a. actually accomplished: I practiced PCR all by myself and I watched Joey perform
qPCR.
b. concerns: I should really start on my proposal!!!
c. learning (workplace, science, project, yourself): I learned that Borrelia is NOT
Rickettsia even though I'm working in the Rickettsial Department. I also learned about
ELISA. I had a basic understanding of qPCR.
4. Planning
I hope to do PCR more flawlessly tomorrow and perform a qPCR all by myself. I also
hope to analyze the ClustalW Borrelia sequences with Dr. Ju.
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