DNA swarm robots by team Handai
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Experiment

In general, the experiments involves deciding annealing condition and mostly observation. Firstly, the appropriate annealing condition concerning temperature, buffer composition, and time was determined. Then, gel electrophoresis was performed to approximate if the structure has annealed or not and finally atomic force microscopy (AFM) was used to analyze the structures. Finally, additional procedures were also performed for additional mechanisms implemented in the structure.


  • Annealing
    • Reagents
    • Procedure
  • Gel electrophoresis
    • Reagents
    • Procedure

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  • Annealing
    • Reagents
    • Procedure
  • Gel electrophoresis
    • Reagents
    • Procedure

    • Back to top

Determination of optimal annealing condition

Reagents

DNA origami

Reagents Volume (µl)
1 Staple mix 26
2 M13MP18 Scaffold 52
3 Purified water 39
4 10X buffer (refer to next table) 13

10X Buffer

Reagents Volume (µl)
1 1M Tris 25
2 0.5M EDTA 10
3 2.5M NaCl 10
4 2.5M MgCl2 20
5 Purified water 435

Procedures

  1. Dispense the reagents as stated in the tables above into a PCR tube .
  2. Anneal the reagents in the PCR tube using TaKaRa PCR Thermal Cycler Dice® Touch

Gel electrophoresis

Gel electrophoresis was performed after every annealing process and before observing through AFM. Gel electrophoresis itself involves flowing the annealed DNA materials through an electric field in gel. As DNA has a phosphate group that is negatively charged, the DNA origami will flow through the gel from the negative electrode to the positive electrode. In the process, smaller pieces will be able to flow through the gel quicker than bigger pieces or possibly the pieces have properly folded.

Reagents

Gel

Reagents Volume/Mass
1 10X TBE 2.5 ml
2 1M MgCl2 0.65 ml
3 Agarose 0.5 mg
4 Purified water 46.85 ml

Buffer

Reagents Volume/Mass
1 10X TBE 2.5 ml
2 1M MgCl2 1 ml
3 Purified water 46.5 ml

Procedures

    Gel preparation

  1. Mix all the reagents together into a 100 ml conical flask.
  2. Heat the reagents in microwave until it boils.
  3. Cool the hot mixture in the refrigerator for about 15 minutes.
  4. After 15 minutes, the gel mixture was poured into the gel board. Combs were also inserted to create wells.
  5. The cooled gel was then put in the electrophoresis chamber along with sufficient amount of buffer solution.

    6. Sample preparation

  6. transfer 5 µl of the annealed DNA origami from the annealing tube to a pcr tube.
  7. 3 µl of Rod LD (staining agent) was then added to each tube to form a total 8 µl of solution.
  8. 10 µl of marker was also transferred to one of the wells for comparison.

    9. Running the gel

  9. After setting up the samples, the electrophoresis was set to run at 100 volts for 1 hour
  10. The finished gel was then transferred with another container containing the buffer and 4 µl of SYBR safe (intercalating agent) was pipetted onto the gel.
  11. The gel was shaken on a shaker machine slowly for around 10 minutes.
  12. Image of the gel was finally taken using gel photo machine.

Purification of annealed sample

Upon confirming the annealing results using electrophoresis, the results were firstly purified using column in the centrifuge before incubation for hybridization reactions and before AFM observation. This step was performed to prevent the aggregation of excess materials that might interfere in hybridization experiments and also to prevent the excess materials from interfering and causing irrelevant noise during AFM observation.

Materials

  1. Illustra MicroSpin column S-300
  2. Annealed sample

Procedures

  1. 1. Wash the column with buffer twice. Centrifuging at 700g for 1 minute for each washing.
  2. 2. Dispense the sample into the column.
  3. 3. Centrifuge at 700g for 1 minute.
  4. 4. Collect the eluate for observation.