Biomod/2013/Komaba/Experience

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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)

ConcentrationAmount Stock
Mg2+ 12.5mM 1.25μL 1M
TAE10x 10μL
M13 2.5nM 6.25μL 40nM
Staple Mix25nM 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.

Image:201309191156.jpg

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.

Image:201309201508.jpg


  • This is the image of the Ring.

Image:201309201639(ring).jpg



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
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