Biomod/2013/Komaba/Experiment

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(September 13th  Observing Cylinder and Ring(first ver.) by AFM)
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[[Image:201309191156 (640x558) (2).jpg|frame|Ring 15-equivalent]]
[[Image:201309191156 (640x558) (2).jpg|frame|Ring 15-equivalent]]
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<p>There might have been artificial mistakes, we decided to make the staple mix from the first.
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<p>There might have been artificial mistakes. Thus, we decided to make the staple mix from the first.
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===September 13th  Agarose Gel Electrophoresis(Cylinder and Ring)===
===September 13th  Agarose Gel Electrophoresis(Cylinder and Ring)===
<p>Since we failed to observe our origami by AFM, we decided to try electrophoresis.<br>
<p>Since we failed to observe our origami by AFM, we decided to try electrophoresis.<br>

Revision as of 07:35, 26 October 2013


Contents

Overall

 To make our DNA Screw, we needed a Cylinder and a Ring made of DNA Origami. The structure was observed by AFM. The final structure was made from a single scaffold strand so that the electrostatic interaction between the Cylinder and the Ring could be avoided, but in order to make sure we could make the structre of the cylinder and the ring, at first, we made a cylinder and a ring separately. Although the image of the Ring was not something we had expected but the we succeeded in making the Cylinder. Second, we made the final structure of the Cylinder and the Ring. The structure was made from a single scaffold strand, which means there were connected to each other. We observed the structure by AFM and we could see the structure! Third, we synthesized the spiders and attached them on the cylinder but unfortunately, we couldn't observe what we had expected by AFM.

Method and Result

Cylinder(first version)

We made a Cyliner by DNA Origami.

  • First, we made staple mix for the Cylinder. We put 2.5μL for each staple whose stock is 50μM into the tube and diluted to 250nM with TE buffer. We had a set of 179 staples and we had total amount of 500μL. After putting all the protocols into the tube, we vortexed for a few seconds.
  • Second, we made 100μL solution of the Cylinder. We made 10-equivalent and 15-equivalent. As for 10-equivalent, we put 1.25μL of Mg2+(stock concentration: 1M), 10μL of TAE 10x, 6.25μL of M13mp18 (stock concentration: 40nM), 10μL of Staple Mix (stock concentration: 250nM), and 72.5μL of mQ into the tube. As for 15-equivalent, we put 1.25μL of Mg2+(stock concentration: 1M), 10μL of TAE 10x, 6.25μL of M13 (stock concentration: 40nM), 15μL of Staple mix (stock concentration: 250nM), and 67.5μL of mQ. After putting all the protocols into the tube, we vortexed for a few seconds.
  • Third, we put the tube into an annealing machine. We annealed it from 90°C to 20°C -0.2°C/min.

  • Here, we put the table of amount, final concentration and stock concentration of each protocols.

    Staple Mix

    AmountStock
    Staple2.5μL(each)50μM
    TE buffer52.5μL
    Total500μL


    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



    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


    Ring (first version)

    We made a Ring by DNA Origami.

    • First, we made staple mix for the Ring. We put 5μL for each staple whose stock is 50μM into the tube and diluted to 500nM with TE buffer. We had a set of 80 staples and we had total amount of 500μL. After putting all the protocols into the tube, we vortexed for a few seconds.
    • Second, we made 100μL solution of the Ring. We made 10-equivalent and 15-equivalent. As for 10-equivalent, we put 1.25μL of Mg2+(stock concentration: 1M), 10μL of TAE 10x, 12.5μL of M13mp18 (stock concentration: 40nM), 10μL of Staple Mix (stock concentration: 500nM), and 66.25μL of mQ into the tube. As for 15-equivalent, we put 1.25μL of Mg2+(stock concentration: 1M), 10μL of TAE 10x, 12.5μL of M13 (stock concentration: 40nM), 15μL of Staple mix (stock concentration: 250nM), and 61.25μL of mQ. After putting all the protocols into the tube, we vortexed for a few seconds.
    • Third, we put the tube into an annealing machine. We annealed it from 90°C to 20°C -0.2°C/min.

    • Here, we put the table of amount, final concentration and stock concentration of each protocols.


      Staple Mix

      Amount Stock
      Staple 5μL(each) 50μM
      TE buffer 100μL
      Total 500μL


      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

      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

      Observing Cylinder and Ring(first ver.) by AFM

      Ring 15-equivalent
      Ring 15-equivalent

      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 the images of our samples which we made.


      There might have been artificial mistakes, so we decided to make the staple mix from the first.




      Lab Notes

      We conducted our experiments at Fuji Laboratory, Komaba Research Campus.

      September 11th  Cylinder and Ring(first ver.)

      Staple Mix (Cylinder)

      AmountStock
      Staple2.5μL(each)50μM
      TE buffer52.5μL
      Total500μL

      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.

      Ring 15-equivalent
      Ring 15-equivalent

      There might have been artificial mistakes. Thus, we decided to make the staple mix from the first.


      September 13th  Agarose Gel Electrophoresis(Cylinder and Ring)

      Since we failed to 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 on 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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