Biomod/2013/Waterloo

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Contents

Video

Check out our YouTube video! [1]

Assembly of the Walker

  • 1M Tris-HCl, pH 7.5
  • 0.5M Acetic Acid
  • 100mM EDTA
  • 100mM Magnesium Acetate
  • DNA Working Stocks (~1uM): Walker-{1-7}
  • 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:
    1. Add 580 uL millipore water to the microcentrifuge tube.
    2. Add 188 uL of 100mM Magnesium Acetate to the PCR tube.
    3. Add 60 uL of 1M Tris-HCl to the PCR tube.
    4. Add 60 uL of 500 mM Acetic Acid to the PCR tube.
    5. Add 37.5 uL of 100 mM EDTA to the PCR tube.
    6. Vortex the microcentrifuge tube for 30 seconds.
    7. 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:
    1. Cassette One: 1SP1 - 5.6 uL, 1SP2 - 4.5 uL
    2. Cassette Two: 2SP1 - 6.6 uL, 2SP2 - 4.5 uL
    3. Cassette Three: 3SP1R2 - 4.6 uL, 3SP2R2 - 6.1 uL
  13. Stir the mixture with a pipette for 5 minutes, then let the solution sit for six hours.
  14. Freeze!!!

Results

DNA Tiles

Description This is a transmission electron microscopy image of our tile. This image shows that we successfully created our DNA tiles. They are the most complex structure in the system and provide strong evidence that the other structure, too small to image but much less complex, were also created. Image:Tile1.png

Description Supporting evidence similar to the image above.

Image:Tile2.png

Gel Electrophoresis Results

On July 18th, 2013 we performed Experiment #43 where we ran our first non-denaturing polyacrylamide gel electrophoresis. The purpose of this experiment was to determine the quantity of DNA origami Walkers that formed in solution over various synthesis conditions. In preparation for this experiment, five Walker samples were created over the course of seven days. Each of these samples were diluted to the same concentration of 50 nM, ramped to 70°C at a rate of 0.1°C/second and held at 70°C for 10 minutes. Each sample was cooled from 70°C to 25°C but the duration of the cool down differed between samples. By running the non-denaturing polyacrylamide gel electrophoresis, we could determine what condition gave the best results and what had to be done next to achieve even greater results. The results of uwDNA Experiment #43 are shown in Figure 1 below.

Figure 1

Image:WaterlooGel1.png

Each vertical line – or smear – you see corresponds to a certain walker sample that was run. And each horizontal line – we will call them bands – represents a certain substance of similar molecular weight in our solution, all bands along the same horazontal are the same. The intensity of the band is representative of its concentration. Starting from the left, the Walker samples follow: Experiment #11, Experiment #12, Experiment #13, Experiment #15, Experiment #17. Each sample were created using the same DNA strands at the same concentration with the same buffers and had the same heating ramp and holding time, they differ only by their cool down time. Starting from the left, to list these alterations:

  • Experiment #11: 20 minutes to cool from 70°C to 25°C
  • Experiment #12: 12 minutes to cool from 70°C to 25°C
  • Experiment #13: 90 minutes to cool from 70°C to 25°C
  • Experiment #15: 180 minutes to cool from 70°C to 25°C
  • Experiment #17: 780 minutes to cool from 70°C to 25°C

As we can see, the results are not great. For each sample we have four distinct bands and a smear that is present throughout the all of the samples. We suspect that the first set of bands along the same horizontal are our Walkers and the next three bands are unbound strands, contamination, or secondary DNA complexes. There are three very faint bands on top of the Walker bands present in samples 11, 13, and 17, these are suspected to be multi-Walker fusions. Upon further analysis of our data, we can see that there is a direct correlation between cool down time and non-Walker band concentration. We hypothesize that an increase in cool down time will decrease the concentration of non-Walker bands. We can see this by looking at Experiments 11 and 17. Experiment 17 had a 780 minute cool down which resulted in very faint non-Walker bands whereas Experiment 11 had a 20 minute cool down which resulted in very intense non-Walker bands.

To test our hypothesis – cool down time is inversely proportional to the amount undesired products – we decided to run another gel on July 25th, 2013. The purpose of this gel was to attempt to identify what the non-Walker bands found in Experiment #43, and to test purification techniques and increased cooling time in order to optimize our yield. The results of uwDNA Experiment #50 are shown in Figure 2 below.

Starting from the left, our samples follow: DNA Ladder, Experiment #17, Experiment #44, Experiment #46, and Experiment # 45 for the last three wells. The purpose of the DNA ladder was to identify the unknown bands, Experiments number’s 17, 44, and 46 were all Walker samples created under different thermocycling conditions, and Experiment #45 samples were used to test the effectiveness of Walker purification under different conditions. To list these alternations:

  • DNA Ladder: 3 DNA strands of varying molecular weight
  • Experiment #17: 10 minutes held at 70°C, 780 minutes to cool from 70°C to 25°C
  • Experiment #44: 120 minutes held at 70°C, 360 minutes to cool from 70°C to 25°C
  • Experiment #46: 120 minutes held at 70°C, 780 minutes to cool from 70°C to 25°C

All Walker samples in Experiment #45 were held for 60 minutes at 70°C and cool from 70°C – 25°C over 60 minutes. They differ in there purification conditions.

  • Experiment #45 - 1: Milli-Q Rinse, Ethanol Rinse, Buffer Rinse
  • Experiment #45 - 2: Milli-Q Rinse, Buffer Rinse
  • Experiment #45 - 3: No Rinse

By observing the results, we have found all the answers to the questions previously asked. By comparing Experiment numbers 17, 44, and 46 we can confirm that an increase in cool down time as well as an increase in holding time will decrease undesired bands. These undesired bands have also been identified by our DNA ladder. Because we know the exact contents of our DNA ladder, we will know what is in each band that the DNA ladder creates and by comparing this along the horizontal, we can conclude that the non-Walker bands are simply non-bonded Walker DNA strands. The results from this experiments also show us the benefit of purification, the last three samples are all purified Walkers and we can see that the results are significantly cleaner that the unpurified ones. By analyzing the purified samples we can conclude that an ethanol rinse is not required, but a Milli-Q and Buffer are.

This experiment is a great improvement to our understanding of DNA origami synthesis, but it does leave some questions. What is creating the large smears on the top most bands, what exactly are the top bands, and why did the top most bands not move through the gel.

Figure 2

Image:WaterlooGel2.png

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