Biomod/2013/Waterloo

Required Materials

1. 1M Tris-HCl, pH 7.5
2. 0.5M Acetic Acid
3. 100mM EDTA
4. 100mM Magnesium Acetate
5. DNA Working Stocks (~1uM): Walker-{1-7}
6. 1x 200uL PCR tube

Procedure

1. Remove DNA working stocks from the freezer and allow them to thaw
2. Prepare the buffer:
3. Add 49.5 uL of milli-Q water to the PCR tube
4. Add 12.5 uL (6+6.5uL) of 100mM Magnesium Acetate to the PCR tube
5. Add 4 uL of 1M Tris-HCl to the PCR tube
6. Add 4 uL of 500 mM Acetic Acid to the PCR tube
7. Add 2.5 uL of 100 mM EDTA to the PCR tube
8. Vortex the PCR tube for 30 seconds
9. Determine A260 of each strand using the spectrophotometer using Oligocalc (http://www.basic.northwestern.edu/biotools/OligoCalc.html) and the sequence of the strand, determine the concentration of the strand. The concentration of each strand in the PCR tube must be 50nM. Add a volume of the DNA working stock to the PCR tube equal to the table below.
10. Close the PCR tube, tap down any droplets that may be on the sides of the container and vortex for one minute
11. Open the PCR machine and place the PCR tube inside any of the compartments
12. Close the PCR machine, go to the computer and run qCycle.exe
13. Press the Load/Edit button and open the appropriate walker synthesis script, depending on the cooling duration requirements for that trial
14. Press Start Program
15. Enter 100 uL into the text box when prompted for the solution volume
16. Allow the PCR machine to run. This process will take a variable amount of time, depending on the length of the cooling process.

Strand-ID	Volume (μL)
Walker 1	3.9
Walker 2	5.3
Walker 3	4.3
Walker 4	3.3
Walker 5	3.9
Walker 6	3.9
Walker 6	2.9

Assembly of the Cassettes

The cassettes are much more complex than the walker, as they are made up of many more strands.

Only one cassette should be assembled at a time within each PCR tube.

Required Materials

• 1M Tris-HCl, pH 7.5
• 0.5M Acetic Acid
• 100mM EDTA
• 100mM Magnesium Acetate
• DNA Working Stocks (~1uM)
• 1x 200uL PCR tube

Procedure

1. Remove DNA working stocks from the freezer and allow them to thaw
2. Prepare the buffer:
3. Add milli-Q water to the PCR tube:
4. 11.2 uL for Cassette 1. 8.1 uL for Cassette 2. 10.2 uL for Cassette 3
5. Add 25 uL of 100mM Magnesium Acetate to the PCR tube
6. Add 8 uL of 1M Tris-HCl to the PCR tube
7. Add 8 uL of 500 mM Acetic Acid to the PCR tube
8. Add 5 uL of 100 mM EDTA to the PCR tube
9. Vortex the PCR tube for 30 seconds
10. Determine A260 of each strand using the spectrophotometer.
11. Using Oligocalc (http://www.basic.northwestern.edu/biotools/OligoCalc.html) and the sequence of the strand found, determine the concentration of the strand. The concentration of each strand in the PCR tube must be 50nM. Add a volume of the DNA working stock to the PCR tube equal to the previously calculated values.
12. Close the PCR tube and vortex for one minute
13. Open the PCR machine and place the PCR tube inside any of the compartments
14. Close the PCR machine, go to the computer and run qCycle.exe
15. Press the Load/Edit button and open the appropriate cassette synthesis script, depending on the cooling duration requirements for that trial
16. Press Start Program
17. Enter 150 uL into the text box when prompted for the solution volume
18. Allow the PCR machine to run. This process will a variable amount of time, depending on the requirements for that trial

Formation and Purification of Tile

This is the largest DNA structure.

Required Materials

1. Remove the M13 genomic DNA from the freezer to allow it to thaw
2. Remove DNA working stocks from the freezer and allow them to thaw
3. Prepare the buffer: Add 580 uL millipore water to the microcentrifuge tube. Add 188 uL of 100mM Magnesium Acetate to the PCR tube. Add 60 uL of 1M Tris-HCl to the PCR tube. Add 60 uL of 500 mM Acetic Acid to the PCR tube. Add 37.5 uL of 100 mM EDTA to the PCR tube. Vortex the microcentrifuge tube for 30 seconds. Determine A260 of each strand using the spectrophotometer
4. Using Oligocalc and the sequence of the strand, determine the concentration of the strand. The concentration of each staple strand in the microcentrifuge tube must be 10.5 nM, and the staple strands with extensions should be 3.0 nM. Add a volume of the DNA working stock to the microcentrifuge tube equal to the table found here.
5. Add 5 uL of the 1ug/uL M13 DNA solution.
6. Close the microcentrifuge tube and vortex for one minute
7. Divide the contents of the microcentrifuge tube into 10 PCR tubes by adding 150 uL of the solution in the microcentrifuge tube into each PCR tube
8. Open the PCR machine and place the PCR tubes inside any of the compartments
9. Close the PCR machine, go to the computer and run qCycle.exe
10. Press the Load/Edit button and open the tile origami synthesis script
11. Press Start Program
12. Enter 150 uL into the text box when prompted for the solution volume
13. Allow the PCR machine to run. This process will take approximately 130 minutes
14. When completed, open the PCR machine, remove the PCR tubes, label and freeze
15. To purify the tile, fill one 100k MWCO spin column with 3 PCR tubes and follow the Purification procedure

Assembly of DNA Devices

Required Materials

• 1x 0.6 mL microcentrifuge tube
• Walker Anchor Strands: A-1,A-2,A-4
• 1M Tris-HCl, pH 7.5
• 0.5M Acetic Acid
• Milli-Q Water
• 100 mM EDTA
• 100 mM Magnesium Acetate
• Desired Walker, Cassette, Tile Solution

Procedure

1. Take a sterile 0.6 mL microcentrifuge tube and label it after the appropriate Assembly Number
2. Depending on the experiment, you will have to thaw out a purified tile, a walker, all three cassettes, anchor strands yA-1, yA-2, and yA-4. Once thawed, vortex each tube for ~2 seconds at 2500 rpm and then place into an ice bath
3. Add 140.55 uL of Milli-Q water into the Assembly tube
4. Add 25 uL of 100 mM Magnesium Acetate into the Assembly tube
5. Add 8 uL of 0.5M Acetic Acid into the Assembly tube
6. Add 8 uL of 1M Tris-HCl into the Assembly tube
7. Add 4 uL of 100 mM EDTA into the Assembly tube
8. Add 4 uL of Walker solution and 8 uL of each cassette into the Assembly tube
9. Add 15.45 uL of Purified Tile into the Assembly tube
10. Add 7.41 uL of yA-1, 2.67 uL of yA-2, 1.82 uL of yA-4 into the Assembly tube
11. The solution should now be ~ 224 uL. Extract 112 uL from the tube and place it into a 200 uL PCR tube. Do this twice
12. Open the lid to the PCR machine and place both PCR tubes anywhere inside
13. Open qCycler.exe on the computer and Load the “Assembly” file
14. Once opened select “Run” and set the volume to 112 uL.
15. Press enter and wait 24 hours
16. Freeze solutions.

Walking

Required Materials

• Walker Anchor/Anchor-Fuel Strands: A-1 to A-9, FA-1 to FA-9
• Fuel-Shield-1, Fuel-Shield-2, and Fuel-Shield-3 Strands
• Assembled Origami Solution

Procedure

1. Before walking take out four PCR tubes and label them: S1, S2, S3,Final. This is for collecting 5 uL samples at each of the three stages of the walking process.
2. Add in __ of each Fuel-Shield-1, Fuel-Shield-2, Fuel-Shield-3 to the PCR tube.
3. Wait for 2 hours
4. Take the S1 PCR tube and extract 5 uL from the solution tube. Freeze immediately.
5. Add 2.53 uL and 3.39 uL of strands FA-1 and FA-4 and then wait two hours
6. Add 1.82 uL of strand A-3 and wait two hours
7. Add 4.88 uL of strand FA-2 and wait two hours
8. Add 9.52 uL and 2.74 uL of strands A-5 and A-6 and wait two hours
9. Transfer 5 uL from the walking solution to the S2 PCR tube. Freeze immediately.
10. Add 6.67 uL and 5.13 uL of strand FA-3 and FA-6 and wait two hours
11. Add 2.9 uL of strand A-7 and wait two hours
12. Add 3.09 uL of strands FA-5 and wait two hours
13. Add 4.26 uL and 5 uL of strands A-8 and A-9 and wait two hours
14. Transfer 5 uL from the walking solution to the S3 PCR tube. Freeze immediately.
15. Add 3.51 uL, 6.67 uL and 8 uL of strands FA-7, FA-8 and FA-9 and wait two hours
16. Transfer 5 uL from the walking solution to the Final PCR tube. Freeze immediately.

Cassette Preprogramming

Required Materials

• Synthesized cassettes #1-3
• Working solution of cassette activation/deactivation strands
• 1M Tris-HCl, pH 7.5
• 0.5M Acetic Acid
• Milli-Q Water
• 100 mM EDTA
• 100 mM Magnesium Acetate

Procedure

1. Cassettes are synthesized in there JX2 form. This means they are initially inactive. To activate them we must add in the corresponding Set-P1/2 and Fuel-P1/2 DNA strands. To deactivate them we must add in the corresponding Set-J1/2 and Fuel-J1/2 DNA strands. For the purposes of this experiment we will be activating all of the cassettes.
2. To do this, take the cassettes from the freezer to thaw. Once thawed briefly vortex at a speed of 2500 rpm for above 1 second, do the vortexing twice. After vortexing, place the PCR tubes into an ice bath.
3. Take three steril 0.6 mL microcentrifuge tubes and label them after each cassette
4. Fill each tube with 100 mL of their corresponding cassette
5. Add 12.5 uL of 0.1M Magnesium Acetate
6. Add 4 uL of 1M Tris-HCl
7. Add 4 uL of 0.5M Acetic Acid
8. Add 2.5 uL of 0.1M EDTA
9. Add 50.9 uL of Milli-Q water into the Activated Cassette #1 tube , 44.9 uL into the Activated Cassette #2 tube, 44.1 uL into the Activated Cassette #3 tube
10. Add in the appropriate Fuel Strands:
    1. Cassette One: 1FJ1 - 6.3 uL, 1FJ2 - 4.7 uL
    2. Cassette Two: 2FJ1 - 5.0 uL, 2FJ2 - 6.7 uL
    3. Cassette Three: 3FJ1 - 4.5 uL, 3FJ2 - 5.3 uL
11. Stir the mixture with a pipette for 5 minutes, then let the solution sit for two hours.
12. Repeat step 3 but this time use the corresponding Set-P1 and Set-P2 strands. Add the corresponding Set volumes:

• Cassette One: 1SP1 - 5.6 uL, 1SP2 - 4.5 uL
• Cassette Two: 2SP1 - 6.6 uL, 2SP2 - 4.5 uL
• Cassette Three: 3SP1R2 - 4.6 uL, 3SP2R2 - 6.1 uL

1. Stir the mixture with a pipette for 5 minutes, then let the solution sit for six hours.
2. Freeze!!!