May 31^st

Gel recovery procedure

• Excise the DNA fragment from the agarose gel.
• Weigh the gel slice and add 3 volumes of Extraction Buffer to 1 volume of gel（100mg=100μl）. The gel slices’ weight were 0.2766g，0.2591g and 0.2121g.
• Incubate at 50℃ until the gel melts in a heating block and vortex the tube every 2-3 minutes during the incubation.
• Apply the sample to Spin column, centrifuge for 1min at 6000xg. Discard the flow-through.
• Add 500μl Extraction Buffer to Spin column, centrifuge for 60s at 12000xg. Discard the flow-through.
• Add 750μl Wash Buffer to Spin column, wait for 3minutes, than centrifuge for 60s at 12000xg. Discard the flow-through.
• Centrifuge for an additional 1 min at 12000xg and transfer the Spin column to a sterile 1.5 ml micro-centrifuge tube.
• Add 50μl elution buffer to the Spin column and let it stand for 1 minute at room temperature.
• Centrifuge for 1min at 12000xg. The Buffer in the microcentrifuge tube contained the DNA.
• The OD260 that measured was 0.120 OD

June 1^st

The first assembly

• Dilute everyone of the 118 staples to a concentration of 100 μM. Take 10μl of each and add 820 μl ddH2O to make the concentration of the staple mixture 500 nM.
• For the Tris-Hcl（Mg²⁺）Buffer, 0.012g Tris base, 0.0268g MgAc and 80μl HCL were added to reach a final concentration of 10mM Tris and 12.5mM Mg²⁺.

The annealing reaction

• The reaction was set as the following table(Scaffold:Staple=1:10)

The reaction was set as the following table(Scaffold:Staple=1:10)

	Volume	Final concentration
Scaffold	15μl	1.7nM
Staple	5μl	17nM
Reaction buffer(Tris)	30μl	6mM Tris, 7.5mM MgAc

• Reaction condition: 50℃ for 1h , stored at 4℃
• The staple mixture (500nM) was diluted into a concentration of 170nM, then added into the reaction tube.

June 5^th

Preparation for the silicon slice

• Heat the slice in a mixture of 35ml Dense H₂SO₄ and 15ml H₂O₂ for 20min, until there are no bubbles
• Discard the solution. Wash the slice with water for several times then put them into a clean beaker.
• Add ultrapure water for liquid sealing and seal the beaker with sealing film.

Sample adsorption

• Take a 5μl volume of the sample, let it be deposited onto the silicon slice.
• Left to adsorb for 5 min.
• Wash with ddH2O to remove the salt, then let it to air dry and wait for imaging.

June 6^th

Characterization of the nanostructure by AFM （Sample from June 1^st）

June 8^th

June 9^th

The annealing reaction

• The reaction was set as the following table(Scaffold:Staple=1:10&1:5)

The reaction was set as the following table(Scaffold:Staple=1:10&1:5)

	Volume	Final concentration
Scaffold	15μl	0.6nM
Staple	5μl	6nM/3nM
Reaction buffer(Tris)	30μl	6mM Tris, 7.5mM MgAc

• Reaction condition: Pre-denaturation at 95℃ for 4 min. Denaturation at 95℃ for 30s, annealing at 60℃ for 30s, extension at 72℃ for 5min, Denaturation at 95℃ for 30s, annealing at 50℃ for 30s, extension at 72℃ for 5min, 4 cycle. Final extension at 72℃ for 5min, 37℃ for 30min and store at 4℃
• The staple mixture (500nM) was diluted into a concentration of 60nM and 30nM, then added into the reaction tube.

June 14^th

The characterization of closed-state structure.

June 30^th

• Dilute Fuel strand and Anti-fuel strand to a concentration of 100μM.
• Take 10μl from each Fuel tube. Mix it with 80μl ddH2O to make a mixture of two different Fuel strands at a concentration of 10μM.
• Dilute the Anti-fuel strands in the same way.

July 5^th

• Use the newly designed primer to perform an αPCR in the same way we did on May 30^th, except the PCR cycle was changed into 40 times.
• Analyze the products by agarose gel electrophoresis（0.8%）
• Repeat the gel recovery as the procedure on May 31^st.
• The OD260 that measured was 0.109 OD.
• Repeat the annealing reaction in the same procedure as the procedure on June 9^th.

July 11^st

Regulation

• Dilute the Fuel strand mixture to a concentration of 9nM.
• The scaffold was mixed with the newly diluted Fuel solution by 1:1( 15μl each).
• Perform an annealing reaction in a condition of 37℃ for 30min.

July 12^th

Characterization of the nanostructure by AFM(sample from July 9^th)

July 14^th

50× TAE buffer

• 242g Tris, 18.612g EDTA, 57.1ml Glacial Acetic Acid and 800ml deionized water were added to a beaker.
• Its PH was adjusted to 8.3 with NaOH.

10× Mg²⁺ buffer(120mM)

• 0. 1143g MgCl₂ were added to 10ml ddH2O.

The annealing reaction

• Reaction condition: lower the temperature form 80℃ to 40℃ at 4min/2℃, maintain 37℃ for 30min，store at 4℃.

July 15^th

Regulation

• Dilute the Fuel strand mixture to a concentration of 48.75nM.(Diluted with 1× TAE).
• The scaffold was mixed with the newly diluted Fuel solution by 1:1(15μl each).
• Perform an annealing reaction in a condition of 37℃ for 30min, then the temperature slowly decrease to 16℃ at a rate of 3℃/min
• Mix the Fuel+ products with Anti-fuel strand (5nM) by 1:1(15μl each).
• Repeat the third step.

July 18^th

The characterization of open-state structure.

July 21^st

The annealing reaction

• Repeat the annealing reaction in the same procedure on June 9^th

Preparation for TEM

• Drop 4μl of the sample solution on the grid, left to adsorb for 1 min.
• Wash away the excess salt by a drop of ddH2O, then touch the edge of the grid with a filter paper to wick away excess water.
• Touch the grid with a drop of 5% Phosphotungstic acid solution.
• Remove it quickly and touch with a second drop for 30s.
• Let the grid dry and keep it at room temperature.
• Analyze the Fuel+ and Anti+ products by agarose gel electrophoresis (2%)

July 25^th

The characterization of open-state structure.

July 26^th

• Perform an αPCR in the same way that did on July 5^th
• Recover the products by repeating the steps on May 31^st, except for the Elution buffer was changed into ddH2O.
• The gel slices’ weight were 0.2445g, 0.1679g, 0.1413g.
• The OD260 that measured was 0.480 OD.

July 28^th

The characterization of closed-state structure.

July 31^st

The characterization of closed-state structure.

The first step of TMV assembly

• 0.1654g NaH₂PO₄•2H₂O, 6.9237g Na₂HPO₄•12H₂O were added to 100ml ddH2O.(PBS buffer, PH 8.0).
• Dialyse the TMV protein at 4℃ in PBS buffer（PH 8.0）for 2 days.

August 2^nd

The second step of TMV assembly

• 1.22g NaH₂PO₄•2H₂O, 4.37g Na₂HPO₄•12H₂O were added to 100ml ddH2O.(PBS buffer, PH 7.0).
• Change the buffer for dialysis into PBS buffer(PH 7.0).
• Dialyse at 4℃ for 1day.

August 24^th

The annealing reaction

• Reaction condition

80℃→70℃   4min/2℃ 64℃→48℃   4min/2℃ 46℃→40℃   4min/2℃

• Take 10μl the NEB single strand M13 as the scaffold.

August 26^th

DLS

• The sample temperature was maintained at 25℃ during measurement.
• The sample was measured at a concentration of 10 nM.

August 29^th

The characterization of TMV assembly and closed-state DNA Origami

August 31^st

Characterization of the nanostructure by AFM (sample from August 24^th)

September 4^th

The characterization of TMV assembly and closed-state DNA Origami

September 15^th

The annealing reaction

September 18^th

The characterization of TMV assembly and open-state DNA Origami

September 23^th

The first step of TMV assembly

• 0.2481g NaH₂PO₄•2H₂O, 10.3856g Na₂HPO₄•12H₂O were added to 150ml ddH₂O.(PBS buffer, PH 8.0).
• Dialyse the TMV protein at 4℃ in PBS buffer（PH 8.0）for 2 days. September 25th

September 25^th

The annealing reaction

• Reaction condition

80℃→70℃   4min/2℃ 64℃→48℃   4min/2℃ 46℃→40℃   4min/2℃

37℃   30min 4℃   ∞

The second step of TMV assembly

• 1.83g NaH₂PO₄•2H₂O, 6.555g Na₂HPO₄•12H₂O were added to 150ml ddH₂O. (PBS buffer, PH 7.0).
• Change the buffer for dialysis into PBS buffer (PH 7.0).
• Dialyse at 4℃ more than one day.

September 26^th

• Analyze the assembly through agarose gel electrophoresis (2%)

September 27^th

The annealing reaction

September 29^th

The characterization of open-state and closed-state DNA Origami