Biomod/2013/Komaba/Experience
Home,Project,Design,Experiment,Supplementation,Team,Sponsors
Experiments
Cylinder
The Cylinder was made of DNA Origami. It was designed in two dimensions using cadnano 4, and became three dimentional structure after mixing m13, staple mix, TAE 10x, and mQ, and annealing. Staple mix consists of a set 179 strands and TE buffer. The structure of the Cylinder was based on some papers in the "Single-Step Rapid Assembly of DNA Origami Nanostructures for Addressable Nanoscale Bioreactors" by Yanming Fu et al.
Ring
Spider
Lab Notes
We conducted our experiment at Fuji Laboratory, Komaba Research Campus.
September 11th Cylinder and Ring(first ver.)
Staple Mix (Cylinder)
Amount | Stock | ||
---|---|---|---|
Staple | 2.5μL(each) | 50μM | |
TE buffer | 52.5μL | ||
Total | 500μL |
Stock of the staples are 50μM.
There were 179 strands for the Cylinder.
Cylinder(10 - equivalent)
Concentration | Amount | Stock | |
---|---|---|---|
Mg2+ | 12.5mM | 1.25μL | 1M |
TAE10x | 10μL | ||
M13 | 2.5nM | 6.25μL | 40nM |
Staple Mix | 25nM | 10μL | 250nM |
mQ | 72.5μL | ||
Total | 100μL |
Cylinder(15 - equivalent)
Concentration | Amount | Stock | |
---|---|---|---|
Mg2+ | 12.5mM | 1.25μL | 1M |
TAE10x | 10μL | ||
M13 | 2.5nM | 6.25μL | 40nM |
Staple Mix | 37.5nM | 15μL | 250nM |
mQ | 67.5μL | ||
Total | 100μL |
Staple Mix (Ring)
Amount | Stock | ||
---|---|---|---|
Staple | 5μL(each) | 50μM | |
TE buffer | 100μL | ||
Total | 500μL |
Stock of the staples are 50μM.
There were 80 strands for the Ring.
Ring(10- equivalent)
Concentration | Amount | Stock | |
---|---|---|---|
Mg2+ | 12.5mM | 1.25μL | 1M |
TAE10x | 10μL | ||
M13 | 5nM | 12.5μL | 40nM |
Staple Mix | 50nM | 10μL | 500nM |
mQ | 66.25μL | ||
Total | 100μL |
Ring(15 - equivalent)
Concentration | Amount | Stock | |
---|---|---|---|
Mg2+ | 12.5mM | 1.25μL | 1M |
TAE10x | 10μL | ||
M13 | 5nM | 12.5μL | 40nM |
Staple Mix | 75nM | 15μL | 500nM |
mQ | 61.25μL | ||
Total | 100μL |
September 13th Observing Cylinder and Ring(first ver.) by AFM
This is the image of the Ring( 15- equivalent).
Unfortunately, since the AFM was out of control for a while, we couldn't take all all the images of our samples which we made on September 11th.
There might have been artificial mistakes, we decided to make the staple mix from the first.
September 13th Agarose Gel Electrophoresis(Cylinder and Ring)
Since we failed observe our origami by AFM, we decided to try electrophoresis.
Preparing Gel
Concentration | Amount | |
---|---|---|
Agarose | 1.75%(wt/V) | 0.525g |
TBE10x | 1x | 3mL |
Mg2+ | 12.5mM | 375μL |
mQ | up to 30mL | |
Total | 30mL |
- Pour the gel into frames.
- Put frames into a refrigerator.
Preparing Sample
- For each sample, we mix 2μL of loading buffer and 10μL of DNA sample.
We prepared seven samples - Cylinder(10 - equivalent), Cylinder(15 - equivalent), Cylinder (Staple mix), M13, Ring (Staple mix), Ring(15 - equivalent), Ring(10 - equivalent). - Put seven samples on the gel and pour TBE buffer into the container.
- Leave 1.5 hours under 50V condition.
Observing
We tried to observe this using SyBR Green 1 Dye, but we couldn't to see anything separated. The condition of the gel might not have been good.
September 19th Cylinder and Ring(first ver.)
We retried creating samples of our first version of Cylinder and Ring under exactly the same condition as September 11th.
September 20th Observing Cylinder and Ring(first ver.) by AFM
- This is the image of the Cylinder.
- This is the image of the Ring.
As you can see, we succeeded in making the Cylinder but we failed to make the Ring.
After a careful consideration, we found out that the design of the structure was not good. Therefore, we designed a new version of the Ring.
October 15th Ring(third ver.)
Staple
Dilute the oligo to 100μM.
Staple Mix
Concentration | Amount | Stock | |
---|---|---|---|
Staple | 1μM | 2μL | 100μM |
TE buffer | 176μL | ||
Total | 200μL |
There were 12 strands for this Ring.
Ring
Concentration | Amount | Stock | |
---|---|---|---|
Mg2+ | 14mM | 0.7μL | 1M |
TBE 10x | 5μL | ||
Staple Mix | 2.5μM | 25μL | 5μL |
mQ | 19.3μL | ||
Total | 50μL |
Annealing Process
- 90°C~15°C
- -0.1°C/min.
- more than 12 hours