Biomod/2011/TeamJapan/Sendai/Results/Atomic Force Microscope/Observation diary

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observation diary

9/8
We observed 2D structures(2h annealing; M13:staples=1:100) all the day.
The Structure's profile was uncertain.
We guessed the cause was TAE buffer.

9/9
We observed 2D structures(1:100) and DNA origami fields sent from Team Kansai.
Whether we added Nickel to the samples or not, We could see structure's profiles.

9/10
We observed 2D(1:10) and 3D(1:10) structures.
We could see 2D structures as with 1:100.
About 3D structures, we could see something.
However, we didn't make sure they were 3D structure.

9/12
2D structures could be observed condition at 1h annealing.

9/13
In order to remove staples, we centrifuge the obtained structures.
However, only jumbles were observed.
The result suggests that our 3D structures were broken.
From today, Tris(alkalinity) buffer for AFM analyzing was used in substitution for 1×TAE buffer to obtain clear pictures.

9/15
We cut M13.
Our 3D structures could not be observed.

9/20
Observation condition:
over night(room temperature or in refrigerator)
Mg2+(12.5mM, 25mM, 37.5mM)
sumple:field = 1:1, 1:5, 1:10

9/21
We tried to attach our 3D structures to start point on the field on the condition.(oversupply C-legs and start-staples)
However,our 3D structures on the field were not be observed.

9/26
We tried to attach stick to start point on the field on the condition.(oversupply C-leg and start-staple)
However, stick on the field was not observed.

9/28
The 3D structures were annealed on the condition.(37.5mM Mg2+, room temperature)
However, only aggregation was observed.

9/30
The 3D structures were annealed on the condition(25mM Mg2+, room temperture)
However, nothing was observed.
We guessed that we failed annealing.

10/1
We tried to attach stick to the field.
However, stick on the field was not observed.
we experimented to know the most suitable ratio.
However, both 1:100 and 1:1 failed.
We tried to attach spider to the field because we didn't have c-leg.
However, spider on the field was not observed.

10/3
There was no structure after cleaning up with column.

10/4
The 3D structure was cleaned up with column.
The 2D structures were observed before cleaning up with column.
However, only aggregation was observed.

10/7
We observed, 3D structure : field = 1:1, 1:2, 1:3
Only aggregation was observed.

10/11
We observed, 3D strucutre : field(in refrigerator) = 1:3
Only aggregation was observed.

10/13
3D structure on the field was not observed.
We observed field(in refrigerator).
However, the field was not observed.
So we guessed that annealing the field was failed.
3D structure : field(in refrigerator) = 1:1

10/14
3D structure  : field(in refrigerator) = 1:1
We observed 3D structure : field(in refrigerator) = 1:1
However, the field was not observed.
We guessed annealing the field was failed.

10/17
Adjusting C-leg to start-staple was completed.
We observed 3D structure  : field = 1:1
However, 3D structure on the field was not observed.
We observed tri : field = 1:3
The robot was not observed.

10/18
We observed spider+field.
Many fields were observed.
However, spider on the field was not observed.

10/19
We experimented to attach spider to the field.
However, spider on the field was not observed.
We observed new cut 3D structure+field.
However new cut 3D structure on the field was not observed.
We observed cut 3D structure+field using for AFM.
However, 3D structure on the field was not observed.
We observed cut tri2D+field using for AFM.
However, 2D structure on the field was not observed.

10/20
We observed cut 2D structure & the field, 12.5mM at 4°C
There were few fields.
There was no 2D structure.
So we confirmed the only 2D structure again. There were few 2D structures.


10/21
Observation of the 3D structures + fields
Mg2+ 12.5, 15, 20 mM
Temperature 4°C, 25°C
We observed DNA aggregates, but no 3D structures on the fields were observed.


10/22

・12.5mM at room temperature
The field had filled the mica top.
There were few aggregation.
There were few 3D structures.

・15mM at room temperature
The field had filled the mica top.
There were a lot of aggregation and it was difficult to observe.

・12.5mM at 4°C
The field had filled the mica top.
Almost all the fields had broken.
There is no 3D structure.
This is because the day has passed since annealing and the quality of the structure was damaged.

・15mM  at 4°C
The field had filled the mica top.
Almost all the fields had broken.
There is no 3D structure.
This is because the day has passed since annealing and the quality of the structure was damaged.
There were lots of aggregation.

・20mM at 4°C
Almost all the fields had broken.
There is no 3D structure.

・20mM at room temperature
Amount of aggregation is the same as that of (15mM at 4 degree)

10/23
Observation of the sample (3D + field)
Mg2+ 12.5, 15, 20 mM
Temperature 4°C, 25 °C

10/24
Observation of the robots (2D, 3D) and the fields
2D, 3D (M13 : capture-leg = 1 : 5, M13 : Staples = 1 : 10 ratio of concentration)
There were a lot of 2D structures.


10/25
Observation of the sample (2D 3D + field)
2D structure : Field = 2 : 1
3D structure : Field = 2 : 1
We observed the fields, but no 3D structures on the fields were observed.

10/26
Observation of the sample (2D 3D + field)

40 °C, 50 °C
1, 4, 7 hour
There was little difference between 40 °C and 50 °C.


10/28
Observation of the sample (Kansai team)


10/29
Observation of the sample (Kansai team)

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