Sept. 2nd
SEM imaging was reserved at 2 p.m. this Wednesday (Sept. 4th.)
DNA purification was conducted for the “Aug 20th” DNA sample according to the
protocol (see methodology)
Sept. 3th
Follow-up DNA purification was conducted, and the concentration of the purified
DNA sample was determined.
One SEM holder was prepared according to the protocol
Sept. 4th
Results of the DNA purification:
Before
After
DNA concentration 3.4ng/μL 2.2ng/μL
260/280 ratio
1.81
0.98
260/230 ratio
0.01
0.15
According to calculation, the amount of DNA decreased by 75％, and the purity
increased 60％ (i.e. contaminant decreased 60％.)
SEM imaging was performed today. No clear images of quantum dots and DNA
origami was found, hence no images were captured. The next SEM imaging was
reserved next Wednesday (Sept.11th.)
Sept. 5th
The result for the first SEM imaging was discussed. Several things must be noted in
the second trial for SEM imaging:
1. The surface of the carbon tape must be clean, and possible contaminants must be
avoided. A half opened box is used for the air-drying of the sample, and the box
should be stored in the cabinet (demonstrated in the photo below.)
2. Increase the volume of the solution from 20μL to 200 μL to apply on the carbon
tape, so that the whole area of the tape is covered with the solution, which makes
it easier for SEM screening.
One SEM holder was prepared according to the protocol
We decided to perform the 2nd trial DNA purification to confirm the result of the first
purification on Sept. 9th.
Sept. 8th
We held the first meeting of the new semester. During the meeting, we discussed the
proposal for sponsorship from science faculty and schools of Chinese medicine. We
decided to discuss the content of YouTube video next Sunday.
Sept. 9th
A 20mL NaOAc solution (pH=5.2) was prepared
DNA purification was conducted for the “Aug 24th” DNA sample according to the
protocol (see methodology)
Sept. 10th
Follow-up DNA purification was conducted, and the concentration of the purified
DNA sample was determined.
Results of DNA purification:
Before
After
DNA concentration 10.4ng/μL 1.0ng/μL
260/280 ratio
2.00
1.81
260/230 ratio
0.03
0.14
According to calculation, the amount of DNA decreased by 75.96％, and the purity
increased 94.85％ (i.e. contaminant decreased 94.85％.) The purified DNA was then
stored for subsequent assays.
Assuming the molecular weight of the DNA origami is equal to 172 base pairs.
According to the calculation and conversion, a 1.0 ng/μL DNA sample is around
19nM.
The 19nM DNA sample was diluted to 2nM, and DNA-QD conjugation was
conducted in 1nM:1nM ratio for overnight.
The date for FM imaging and AFM imaging was reserved.
Sept. 11th
Results of SEM imaging: no specific pattern was observed on the gold layer, and clear
dots were presented in the gap of the gold layer. However, no significant evidence
showing that the dots were quantum dots.
The next SEM imaging was reserved next Tuesday (Sept. 17th.) After discussion,
since the quantum dot (cadmium selenide) was conductive, gold coating may be
unnecessary for sample preparation. Therefore, gold coating would not be conducted
in the next SEM imaging. It turned out that a control holder with nothing applied
needed to be observed for comparison.
Holder design for Sept. 17th SEM imaging:
Holder
Content
1 Control (clean carbon tape)
2 2nM QD
3 QD-DNA conjugates
Sept. 13th
Today is the deadline for Jamboree registration, submission of project title, and
project abstract. We completed the registration and submission, and we handed in the
proposal for sponsorship to the faculty. We also designed the homepage of our wiki
(not yet uploaded).
15:40 start DNA annealing according to the parameters in August.23rd. Gel extraction
and DNA purification will be performed tomorrow.
Sept. 14th
Gel electrophoresis and DNA extraction was performed.
Results of gel electrophoresis:
:
Results of DNA concentration after gel extraction:
Tube 1
Tube 2
Concentration 5.9 ng/μL 5.3 ng/μL
260/230 ratio
0.01
0.02
DNA purification was conducted according to the protocol (see methodology)
Sept. 15th
Results of DNA purification:
Tube 1
Before
Tube 2
After
Before
After
DNA concentration 5.9 ng/μL 1.3 ng/μL 5.3 ng/μL 2.1 ng/μL
260/230 ratio
0.01
0.18
0.02
0.3
According to calculation, for Tube 1, the amount of DNA decreased by 77.97％, and
the purity increased 98.78％ (i.e. contaminant decreased by 98.78％.) For Tube 2, the
amount of DNA decreased by 60.38% and the purity increased 97.36% (i.e.
contaminant decreased by 97.36 %.)
We held the second meeting of the semester today. In the meeting, we listened to
Yukai to present the rationale of the project in details, brainstormed the scenario of
the YouTube video, and reviewed the judging criteria this year. We arranged a time
next week to take profile photos for the wiki page.
Sept. 17th
The third SEM imaging was conducted.
Results of SEM imaging:
Holder
Content
1 Control (clean carbon tape)
2 2nM QD
3 QD-DNA conjugates
Holder 1 (clean carbon tape), 15.00 kV, 5kX Magnitude
Holder 2 (2nM QD), 15.00 kV, 5kX Magnitude
Holder 3 (QD-DNA conjugates), 15.00 kV, 5kX Magnitude
The background of the control was too fussy that QD and DNA-QD conjugated could
not be distinguished from the background. After discussion, our team decided no
longer using carbon tape as the surface in that carbon tape was not flat enough, which
is not ideal for QD and DNA visualizing. We decided to use freshly-cleaved mica
surface as an alternative for the SEM imaging next time. The next SEM imaging was
reserved next Tuesday (Sept. 24th.)
Sept. 19th
Results of FM imaging:
Clear fibrils were observed in the dye-coated channel (control), but no results
obtained in other channels. Therefore, no images were captured. The result was
presumably caused by the over-incubation for DNA-QD conjugation. We decided to
shorten the incubation time for DNA-QD conjugation from overnight to 15 min,
which thus can be performed right on the day for FM imaging. The next FM imaging
was reserved next Friday (Sept. 27th.)
Sept. 22nd
Due to the Typhoon Usagi, our third weekly meeting was postponed to Sept. 29th.
Sept. 23rd
Three holders were prepared for the SEM imaging next Tuesday:
Mica surface was coated with a thin gold layer prior to the sample settlement.
Holder design for Sept. 24th SEM imaging:
Holder
Content
1 Control (clean mica surface)
2 2nM QD
3 QD-DNA conjugates
Sept. 24th
Results of SEM imaging: clear QD molecules observed.
Holder 1 (clean mica surface), 15.00 kV, 2.5kX Magnitude
Holder 2 (2nM QD), 15.00 kV, 2.5kX Magnitude
Holder 3 (QD-DNA conjugates), 15.00 kV, 350X Magnitude
Sept. 25th
Design for the four channels of FM imaging on Sept. 26th:
STV DNA QD
DNA-QD
Channel 1 -
-
-
-
Channel 2 -
-
-
+ (0.8+20)
Channel 3 +
+
+
-
Channel 4 -
-
+ (10) -
For channel 3, the volume ratio is to be determined on Sept. 26th.
Design for the slides of AFM imaging on Sept. 26th:
STV
DNA
QD
DNA-QD Abeta
1-
-
-
-
-
2-
-
-
+
+
3+
+
+
-
+
4-
-
+
-
+
5-
-
-
-
+
6-
-
-
+
+
7+
+
+
-
+
8-
-
+
-
+
8 glass slides attached with mica surface, 20μL for each slide
4 slides (slide 1 to 4) are prepared today, and the other 4 (slide 5 to 8) will be freshly
made tomorrow.
Results of AFM imaging:
Slide 2 (Abeta + DNA-QD conjugates)
Slide 3 (Abeta + Streptavidin + DNA + QD)
Slide 4 (Abeta + QD)
Slide 5 (Abeta control)
Slide 6 (Abeta + DNA-QD conjugates)
Slide 7 (Abeta + Streptavidin + DNA + QD)
Slide 8 (Abeta + QD)
Fibrils were found in the early-prepared slides but not the freshly-prepared slides;
only dispersed dots were observed in the freshly-prepared slides. Two factors may
contribute to the failure:
(1) The biotinylated Abeta peptides have been degraded;
(2) The way of freshly preparing the samples on the mica surface is not appropriate
The next AFM imaging was reserved next Thursday (Oct.3rd.)
Sept. 26th
Results of FM imaging
STV
DNA
QD
DNA-QD
Channel 1 -
-
-
-
Channel 2 -
-
-
+ (0.8+20uL)
Channel 3 +(1:100, 10uL) +(10uL) +(10uL) Channel 4 -
-
+ (10uL) -
Channel 2: Abeta-DNA-QD
Channel 3: Abeta-STD-DNA-QD
Channel 4: Abeta-QD
According to the results, less abeta fibrils were observed compared with the control
group last Thursday (Sept. 19th), which might be caused by not sufficiently mixed
when diluting the abeta fibrils. Moreover, although Many QD signals were observed,
not any patterns of fibrils were found, which is presumably caused by the effects of
non-specific binding. Therefore, blocking process using BSA solution is suggested for
the FM imaging next time. The next FM imaging was reserved next Thursday
(Oct.3rd.)
Sept. 29th
We held the fourth meeting. Prior to the meeting, we took individual profile photos
with the help of Jessica Wu. In the meeting, we discussed the detailed scenario of the
YouTube video, the results of the AFM, FM, and SEM imaging in the past two weeks,
and we casted a thought for potential applications of the functionalized DNA origami.
Sept. 30th
20:30 Four holders of SEM imaging were prepared as follow:
Holder
Content
1 Control (clean mica surface)
2 2nM QD
3 QD-DNA conjugates (2nM:1nM)
4 QD-DNA conjugates (2nM:0.5nM)