June. 24th
We contacted Dr. Lai, W.C., King and Mr. Fang Yuqiang at City University of Hong
Kong about using atomic force microscope.
June. 25th
We calculated and made 100mL DNA folding buffer (20X DFB) according to the
protocol (see methodology)
June. 27th
We conducted the first DNA annealing by mixing the scaffolds and staples with the
ratio shown below:
Final concentration
Scaffolds 6, 7, B8, 9, 10 100 nM
Staples
1, 2, 3, 4, 5
400 nM
Volume (µL)
20X DFB
2.5
Scaffolds
Staples
6
0.5
7
0.5
B8
0.5
9
0.5
10
0.5
1
2
2
2
3
2
4
2
5
2
RNase free water
35.0
Total
50.0
For details of scaffolds and staples strands, see the methodology for reference.
The condition of temperature is shown below:
Temperature (℃) Cycle
Time
83
1 5 minutes
-1
17 5 minutes
65
1 30 minutes
-1
35 30 minutes
4
1 forever
Total time 19hrs 30mins
The first DNA annealing is started at 17:45, and will end at 13:15 tomorrow
(June.28th)
June. 28th
Result of the first DNA annealing:
The program of the annealing machine was set incorrectly yesterday, so the annealing
ceased early in advance. After discussion, we decided to reset the annealing condition
and conduct the DNA annealing again tomorrow, with the concentration of scaffolds
raised.
June. 29th
We conducted the second annealing by mixing the scaffolds and staples with the ratio
shown below:
Scaffolds
6, 7, B8, 9, 10
200nM
Staples
1, 2, 3, 4, 5
400nM
Volume (µL)
20X DFB
2.5
Scaffolds
Staples
6
1
7
1
B8
1
9
1
10
1
1
2
2
2
3
2
4
2
5
2
RNase free water
32.5
Total
50.0
The condition of temperature, after adjustment, is shown below:
Temperature (℃) Cycle
85
Time
1 5 minutes
-1
21 5 minutes
65
1 30 minutes
-1
41 30 minutes
4
1 Forever
Total time 23 hours
Image of the annealing condition (after adjustment):
A 2% agarose gel electrophoresis was conducted. Photo was captured, and the target
sequence was excised and undergone gel extraction according to the protocol (see
methodology). After gel extraction, the concentration of DNA was determined by
Nanodrop:
DNA concentration 8.6ng/μL
260/280 ratio
1.72
260/230 ratio
0.01
Image of the gel electrophoresis:
June. 30th
A 2% agarose gel electrophoresis was conducted. Photo was captured, and the target
sequence was excised and undergone gel extraction according to the protocol (see
methodology). After gel extraction, the concentration of DNA was determined by
Nanodrop:
DNA concentration 7.4ng/μL
260/280 ratio
3.78
260/230 ratio
0.01
Image of the gel electrophoresis:
After discussion, we decided that since the yielding was quite low, we would explore
the effects of annealing under different concentrations of MgCl2 solution. We will
make 10mM, 20mM, and 30mM MgCl2 tomorrow start the third annealing tomorrow.