1. Mix the following component
Nanomuscle Synthesis
- Optimize the condition of folding DNA origami – MgCl2 concentration.
2. Run the following procedure by PCR machine
(i) heat up to 80°C; incubate for 10 mins
(ii) 80°C to 61°C at 4 min / °C
(iii) 60°C to 25°C at 20 min / °C
​
3. Run gel electrophoresis to find out the optimal concentration of MgCl2
​
​
1. Mix the following components
​
- Optimize the condition of folding DNA origami – the mole ratio between scaffold and staple.
1. Mix the following component
2. Run the following procedure by PCR machine
(i) heat up to 80°C; incubate for 10 mins
(ii) 80°C to 61°C at 4 min / °C
(iii) 60°C to 25°C at 20 min / °C
​
​
- The Folding of DNA origami - Big monomer
Materials and Methods
2. Run the following procedure by PCR machine
(i) heat up to 80°C; incubate for 10 mins
(ii) 80°C to 61°C at 4 min / °C
(iii) 60°C to 25°C at 20 min / °C
​
​
2. Run the following procedure by PCR machine
(i) heat up to 80°C; incubate for 10 mins
(ii) 80°C to 61°C at 4 min / °C
(iii) 60°C to 25°C at 20 min / °C
​
​
1. Mix the following component
-The Folding of DNA origami - Small monomer
Amicon Ultra-0.5 mL Centrifugal Filters are used to remove the excess staples. The following protocol refers to the handbook of the product.
​
​
-
Assemble the filter and the centrifuge tube, load 200μl 1x TE buffer with 10mM MgCl2 into the filter.
-
Spin down at 14000 g for 3 mins.
-
Remove the liquid in the collection tube.
-
Upside down the filter and put back into the collect tube.
-
Spin down at 1000 g for 2 mins.
-
Remove the liquid in the centrifuge tube.
-
Turn the filter back to the original direction, load 200μl of sample in filter.
-
Spin down at 14000 g for 3 mins.
-
Discard the filtrate.
-
Add 200μl of buffer into the filter.
-
Spin down at 14000 g for 3 mins.
-
Discard the filtrate.
-
Upside down the filter and put it into a new collection tube.
-
Spin down at 1000 g for 2 mins.
-
Collect the sample.
- DNA origami Purification
1. Add 50μl of blocking staple solution (400nM) to the big monomer solution ( The mole of blocking staples is five times as the scaffolds).
2. Put the mixture in Mini-Shaking-Hybridisation Oven, and shake at 30 ℃ overnight.
​
​
- Adding blocking staples
1. Mix the blocked big monomer and purified small monomer.
2. Put the mixture in Mini-Shaking-Hybridisation Oven, and shake at 30°C for 3 days.
​
​
- Combination - Big monomer + Small monomer
1. Add 350μl big/small ring-closing-staple solution (400nM) to the dimer solution. (The mole of closing staples is five times as the scaffolds.)
2. Put the mixture in Mini-Shaking-Hybridisation Oven, and shake at 35°C overnight.
​
​
- Closing the ring
1. Add 50μl releasing staples solution (400nM) to the dimer solution (The mole of releasing staple is five times as the scaffolds.)
2. Put the mixture in Mini-Shaking-Hybridisation Oven, and shake at 35°C overnight.
​
- Releasing Big and Small monomer
1. Add 50μl Anti-blocking staples solution (400nM) to the dimer solution. (The mole of Anti-blocking staples are five times as the scaffolds.)
2. Put the mixture in Mini-Shaking-Hybridisation Oven, and shake at 35°C overnight.
​
- Anti-blocking
Analysis
Gel electrophoresis was conducted by a standard procedure as follows.
-
Mix 1×TAE buffer 25 mL and agarose gel powder 0.25 g (1% agarose gel).
-
Heat the mixture by microwave until the powder is completely dissolved.
-
Add 2.5µL of EtBr (1:10000) into the solution and pour the mixture into the plastic case and leave it for 20 minutes at room temperature.
-
Mix 20µL of sample and 4µL of 6X Loading dye.
-
load 20µL of each sample to wells.
-
Run electrophoresis for 25 mins.
-
Observe the gel by UV light (302nm).
​
​
- Agarose Electrophoresis
TEM enables us to see the structures of our products and to measure the exact scales.
Sample preparation
-
Drop 7µL DNA origami solution onto the copper grids.
-
Let the origami settle onto the grid at room temperature for 40 second to 3 mins*.
-
Excess sample is removed by filter paper.
-
The grin then is negatively stained by one drop of 2% uranyl acetate solution
-
Leave it for 40 second to 1 and a half minutes*.
-
Remove the excess dye by filter paper.
-
Air dry for 20 mins.
Observation
The grids are ready for observation. This is performed using an Hitachi H-7650 TEM, at 75kV.
*The time of settlement depends on the concentration of our sample. Every time we purify the product, we will lose part of it, so it requires more time to settle and stain the sample of the final part.
- Transmission electron microscope (TEM)
Operate
- Fuel
- Anti-fuel
1. Add 50μl Fuel staples solution (400nM) to our final structure. (The mole of Fuel staples is five times as the scaffolds.)
2. Put the mixture in Mini-Shaking-Hybridisation Oven, and shake at 35°C overnight.
1. Add 50μl Anti-fuel staples solution (400nM) to our final structure. (The mole of Anti-fuel staples are five times as the scaffolds.)
2. Put the mixture in Mini-Shaking-Hybridisation Oven, and shake at 35°C overnight.
​
