Biomod/2013/Komaba/Experiment

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===Synthesis of DNA spider===
===Synthesis of DNA spider===
The spider was synthesized in the following protocol; 10μL Streptavidin, 11.36μL walking leg, 6.5μL TE, and 6μL NaCl
The spider was synthesized in the following protocol; 10μL Streptavidin, 11.36μL walking leg, 6.5μL TE, and 6μL NaCl
 +
 +
'''Spider'''
 +
<table>
 +
<tr>
 +
<th>
 +
</th><th>Amount
 +
</th><th>Stock
 +
</th></tr>
 +
<tr>
 +
<td>'''Streptavidin'''
 +
</td><td>10μL
 +
</td><td>1.89µM
 +
<tr></tr>
 +
<td>'''walking legs'''
 +
</td><td>11.36μL
 +
</td><td>100μM
 +
</td></tr>
 +
<td>'''NaCl'''
 +
</td><td>6μL
 +
</td><td>
 +
</td></tr>
 +
<tr>
 +
<td>'''TE buffer'''
 +
</td><td>6.5μL
 +
</td><td>
 +
<tr>
===Mixing of Cylinder(first ver.) + common footings and DNA spiders===
===Mixing of Cylinder(first ver.) + common footings and DNA spiders===

Revision as of 00:26, 27 October 2013


Contents

Concept

 To make our DNA Screw, we started creating a Cylinder and a Ring made of DNA Origami. First, we made a cylinder and a ring separately. Every structure was observed by Atomic Force Microscope; AFM. We succeeded in making the Cylinder; however the image of the Ring did not represent the designed structure. Second, we made modified structure of the Cylinder and the Ring. The structure was made from a single scaffold strand, and the Cylinder and the Ring were connected, since the electrostatic interaction between the Cylinder and the Ring could be avoided. We could observe the structure by AFM. Finally, we synthesized the spiders and attached them to the cylinder; however we could not observe expected image by AFM.

Reagents

TAE bufferTris-acetate-EDTA buffer, which is essential to make DNA Origami.
Mg2+Has a function to shape DNA Origami.
TE bufferHas a function to preserve DNA.
TBE bufferTris-Borate-EDTA buffer, which is essential when making DNA tile and doing electrophoresis.
M13mp18Scaffold strand which we bought from TAKARA BIO INC.

Results

Method and Result

Cylinder(1st ver.)

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 thermal cycler. 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 (1st ver.)

    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 Ring(first ver.) by AFM

      Ring 10-equivalent
      Ring 10-equivalent

      This is the image of the Ring( 15- equivalent).
      Unfortunately, since the AFM was out of control for a while, we could only take the image of the Ring (15-equivalent).

      The scan rate was 0.996 Hz.


      Agarose Gel Electrophoresis(Cylinder(1st ver.) and Ring(1st ver.))

      We also tried Gel Electrophoresis to see whether we had only one structure.

      • First, we prepared 30 mL of gel. We put 0.525 % of Agalose, 3 mL of TBE 10x, 375 μL of Mg2+ (stock concentration: 1M) into the tube and diluted with mQ. After adding all the protocols into the tube, we vortexed for a few seconds.
      • Second, we poured the gel into the frame.
      • Third, we put the frame into the refrigerator and left it for 2 hours.
      • Next, we mixed 10 μL of the following protocols and 2 μL of loading buffer for each protocol. The protocols were the solution of Cylinder (10-equivalient), Cylinder (15-equivalent), Ring (10-equivalent), Ring(10-equivalent), Staple mix of Cylinder and the Ring, and M13.
      • Next, we poured 400 μL of TBE buffer into a container and were subjected to electrophoresis at 50 V for one and a half hour at 4°C.
      • Next, we prepared 10 mL solution of SyBR Green 1 Dye. We put 1 μL of SyBR 1 10000x and 99.9 mL of TBE buffer.
      • We moved the gel to the sharre and poured the solution of SyBR.
      • We waited for 1 minute.
      • We put the sharre on the tharmomixer for 10 seconds.
      • We observed under UV light.
      • Here, we put the amount, final concetration, and stock concentration of the protocols we used for the electrophoresis.

        Concentration Amount
        Agarose 1.75%(wt/V) 0.525g
        TBE10x 1x 3mL
        Mg2+ 12.5mM 375μL
        mQ up to 30mL
        Total 30mL


        Unfortunately, the gel did not seperate as we had expected. The concentration of DNA was not high enough to be seperated.

        Cylinder (1st ver.) Retry

        There might have been artificial errors during the last experiment, so we decided to make the Cylinder again. We double checked every step so that we wouldn't make artificial errors.
        We followed exactly the same steps we took in the last experiment, and we used the same amount and kind of protocol as the last experiment.

        Ring (1st ver.) Retry

        There might have been artificial errors during the last experiment, so we decided to make the Ring agin. We double checked evey step so that we wouldn't make artificial errors.
        We followed exactly the same steps we took in the last experiment, and we used the same amount and kind of protocol as the last experiment.

        Observing Cylinder (1st ver.) by AFM

        Cylinder 15-equivalent
        Cylinder 15-equivalent

        This time, we succeeded in observing the Cylinder!

        The scan rate was 0.996 Hz.


        Observing Ring (1st ver.) by AFM

        Ring 10-equivalent
        Ring 10-equivalent

        Unfortunately, the structure was not something we had expected.

        The scan rate was 0.996 Hz.


        Cylinder-Ring

        We made Cylinder-Ring structure with DNA Origami.

      • First, we made staple mix for the Cylinder-Ring. We put 2μL for each staple whose stock is 50μM into the tube and diluted to 200nM with TE buffer. We had a set of 198 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 50μL solution of the Cylinder-Ring. We made 10-equivalent and 15-equivalent. As for 10-equivalent, we put 0.625μL of Mg2+(stock concentration: 1M), 5μL of TAE 10x, 6.25μL of M13mp18 (stock concentration: 40nM), 12.5μL of Staple Mix (stock concentration: 200nM), and 25.625μL of mQ into the tube. As for 15-equivalent, we put 0.625μL of Mg2+(stock concentration: 1M), 5μL of TAE 10x, 6.25μL of M13 (stock concentration: 40nM), 18.75μL of Staple mix (stock concentration: 200nM), and 19.375μL 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 -0.1°C/min.

      • Here, we put the table of the amount, final concentration and stock concentration of protocols used in Cylinder-Ring structure.

        Staple Mix

        Concentration Amount Stock
        Staple 200nM 2μL 50μM
        TE buffer 104μL
        Total 500μL

        10-equivalent

        Concentration Amount Stock
        Mg2+ 12.5mM 0.625μL 1M
        TAE10x 5μL
        M13 5nM 6.26μL 40nM
        Staple Mix 50nM 12.5μL 200nM
        mQ 25.625μL
        Total 50μL

        15-equivalent

        Concentration Amount Stock
        Mg2+ 12.5mM 0.625μL 1M
        TAE10x 5μL
        M13 5nM 6.25μL 40nM
        Staple Mix 75nM 18.75μL 200nM
        mQ 19.375μL
        Total 50μL

        Observing Cylinder-Ring by AFM

        cylinder-ring structure
        cylinder-ring structure

        We successfully observed the cylinder-ring structure. A few cylinders and some ring shaped stractures were seen.

        The scan rate was 0.996 Hz.

        Ring (2nd ver.)

        We made a Ring by DNA Tile just in case.

        • First, we diluted the oligo to 100 μM.
        • Second, we made staple mix for the Ring (2nd ver.). We put 2μL for each staple whose stock is 100μM into the tube and diluted to 1μM with TE buffer. We had a set of 12 staples and we had total amount of 200μL. After putting all the protocols into the tube, we vortexed for a few seconds.
        • Second, we made 50μL solution of the Ring (2nd ver.). We made 10-equivalent and 15-equivalent. As for 10-equivalent, we put 6.25μL of Mg2+(stock concentration: 100mM), 5μL of TAE 10x, 6.25μL of M13mp18 (stock concentration: 40nM), 2.5μL of Staple Mix (stock concentration: 1μM), and 30μL of mQ into the tube. As for 15-equivalent, we put 6.25μL of Mg2+(stock concentration: 100mM), 5μL of TAE 10x, 6.25μL of M13 (stock concentration: 40nM), 3.75μL of Staple mix (stock concentration: 1μM), and 28.75μL of mQ. After putting all the protocols into the tube, we vortexed for a few seconds.
        • Third, we put the tube into an thermal cycler. We annealed it from 90°C to 20°C -0.1°C/min.

        • Here, we put the table of the amount, final concentration and stock concentration of protocols used in Ring (2nd ver.) structure.

          Staple Mix

          Concentration Amount Stock
          Staple 1μM 2μL 100μM
          TE buffer 176μL
          Total 200μL


          10-equivalent

          Concentration Amount Stock
          Mg2+ 12.5mM 6.25μL 100mM
          TBE 10x 5μL
          M13 5nM 6.25μM 40nM
          Staple Mix 50nM 2.5μL 1μL
          mQ 30μL
          Total 50μL


          15-equivalent

          Concentration Amount Stock
          Mg2+ 12.5mM 6.25μL 100mM
          TBE 10x 5μL
          M13 5nM 6.25μM 40nM
          Staple Mix 75nM 3.75μL 1μL
          mQ 28.75μL
          Total 50μL





          Observing Ring (2nd ver.) by AFM

          Ring (2nd ver.)
          Ring (2nd ver.)

          The image was not what we had expected. The cause could could be that we did not need M13 and the annealing time was too short.

          The scan rate was 0.996 Hz.


          Ring (2nd ver.) Retry

          We tried making the Ring (2nd ver.) again. This time, we changed our protocols and annealing time.

          • First, we made staple mix for the Ring (2nd ver.). We put 10μL for each staple whose stock is 100μM into the tube and diluted to 5μM with TE buffer. We had a set of 12 staples and we had total amount of 200μL. After putting all the protocols into the tube, we vortexed for a few seconds.
          • Second, we made 50μL solution of the Ring (2nd ver.). We put 0.7μL of Mg2+(stock concentration: 1M), 5μL of TBE 10x, 25μL of Staple Mix (stock concentration: 5μM), and 19.3μL of mQ into the tube. After putting all the protocols into the tube, we vortexed for a few seconds.
          • Third, we put the tube into an thermal cycler. We annealed it from 90°C to 20°C -0.02°C/min.

          • Here, we put the table of the amount, final concentration and stock concentration of protocols used in Ring (2nd ver.) structure.

            Staple Mix

            Concentration Amount Stock
            Staple 5μM 10μL 100μM
            TE buffer 80μL
            Total 200μL


            Ring (2nd ver.)

            Concentration Amount Stock
            Mg2+ 14mM 0.7μL 1M
            TBE 10x 5μL
            Staple Mix 2.5μM 25μL 5μM
            mQ 19.3μL
            Total 50μL


            Observing Ring (2nd ver.) by AFM

            Ring (2nd ver.)
            Ring (2nd ver.)

            There were not any structures which had similar size of the designed ones.

            The scan rate was 0.996 Hz.


            Cylinder(first ver.) + Common Footings and DNA Spiders with Four Walking Legs

            This part of our experiment aimed at seeing if the walking legs actually work. Here we used DNA spiders with four walking legs, which are different from the DNA spiders described in the Design. We planned to mix common footing,DNA spider, and the cylinder(1st ver.) after the cylinder was synthesized followed by being annealed and the DNA spider was synthesized. Zn2+ starts the DNA spider's cleaving motion. The interval of the footings on cylinder(1st ver.) is too wide for the DNA spider to cross so once all the four legs cleave the occupied footings that they attach, they will be detached from the cylinder. Therefore, taking "before" picture by AFM in which the DNA spiders are attached on the cylinders' surface and "after" picture in which the spiders disappear on the surface are considered to be a proof of the walking legs' function.

            Synthesis of DNA spider

            The spider was synthesized in the following protocol; 10μL Streptavidin, 11.36μL walking leg, 6.5μL TE, and 6μL NaCl

            Spider

            </tr> </tr>

            Mixing of Cylinder(first ver.) + common footings and DNA spiders

            4.0 μl of the solution of the cylinder(1st ver.) which had been annealed for three hours and 16.0 μl of the DNA spiders were mixed into one tube and left it for 30 minutes at room temperature(20~25℃). The solution of the cylinder(1st ver.) which had been annealed for 20 minutes also received the same operation.

            AFM observation

            Spiders
            Spiders


            There were not any structures which had similar size of the designed ones.

            The scan rate was 0.996 Hz.


            Amount Stock
            Streptavidin 10μL 1.89µM
            walking legs 11.36μL 100μM NaCl 6μL
            TE buffer 6.5μL
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