Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Protocols/Origami Purification

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== Origami Purification ==
== Origami Purification ==
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A similar spin protocol appears necessary for binding DNA origami to sticky patches on silicon. Without it, the staples seem to interfere in binding. This protocol was originally taken from Rob Barish's protocol for origami/ribbon constructs. I think that some parts of the protocol may not be necessary for regular origami. Notably, I'm not sure that pipetting up and down is necessary, except perhaps at the last step to maximize recovery. Similarly the use of a 4C centrifuge seems unnecessary, the centrifugation may go much faster at room temperature. If you use a different centrifuge or temperature, calibrate the passage of buffer through the filter before you try purifying any origami to get an idea of how much buffer is retained in the filter after a spin. Fewer spins will be required if a smaller excess of staples is used. You should be able to get away with 10X fewer staples than we typically use according to my standard origami protocol, easily. That is, a 5-10X stoichiometric excess of staples over scaffold is probably fine and will result in cleaner origami faster.
A similar spin protocol appears necessary for binding DNA origami to sticky patches on silicon. Without it, the staples seem to interfere in binding. This protocol was originally taken from Rob Barish's protocol for origami/ribbon constructs. I think that some parts of the protocol may not be necessary for regular origami. Notably, I'm not sure that pipetting up and down is necessary, except perhaps at the last step to maximize recovery. Similarly the use of a 4C centrifuge seems unnecessary, the centrifugation may go much faster at room temperature. If you use a different centrifuge or temperature, calibrate the passage of buffer through the filter before you try purifying any origami to get an idea of how much buffer is retained in the filter after a spin. Fewer spins will be required if a smaller excess of staples is used. You should be able to get away with 10X fewer staples than we typically use according to my standard origami protocol, easily. That is, a 5-10X stoichiometric excess of staples over scaffold is probably fine and will result in cleaner origami faster.
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Wednesday, April 16, 2014

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Origami Purification

Provided by Nadine Dabby and Sungwook Woo

We suggest using the Millipore Microcon YM-100 (blue) spin filters. The times below are estimates for how long to spin your sample (to err on the safe side I would check on the sample after 30 minutes and then estimate how much longer one needs to spin it so as to preserve a small amount of liquid on top of the filter but being VERY careful not to allow it to dry out completely.

this protocol should drastically reduce the presence of excess strands. (the more rounds of adding buffer and filtering you do, the cleaner your sample, but the lower the yield.

Admittedly the yield will be quite low (we haven't figured out exactly how low) -- supposedly each round of filtration should preserve 95% of the origami according to the filter spec but one problem here is dilution (since I have almost always ended up with a sample that is 3X more dilute than I started with). The dilution can be fixed by vaccuum centrifuging at 30 degrees (this does not hurt the origami at all according to Sungwook, but I haven't tried this with our substrate-laden samples.

1. Mix 100uL of your sample with 300uL of 1X buffer of your choice.

2. Vortex mixture and pipette it into a YM-100 spin filter.

3. Spin at 100g for ~30 minutes at 4°C. (Spinning at this low speed prevents the origami from being ripped apart)

4. Remove sample for centrifuge, leaving the machine at 4°C because we’ll be using it again in a moment. The spin filter should have only a few tens of microliters of your sample left in it, just enough to cover the filter. Remove the filter and get rid of any liquid in the tube below. Now reinsert your spin filter and add 200uL of 1X buffer, then pipette up and down just over the filter (five to six times) to recover any bound origami on the filter. Be very careful not to puncture the filter during this step. Finally, add another 200uL of buffer to your sample to bring the final volume to ~400uL.

5. Spin at 100g for ~30 minutes at 4°C.

6. Remove sample for centrifuge, leaving the machine at 4°C because we’ll be using it again in a moment. The spin filter should have only a few tens of microliters of your sample left in it, just enough to cover the filter. Remove the filter and get rid of any liquid in the tube below. Now reinsert your spin filter and add 200uL of 1X buffer, then pipette up and down just over the filter (five to six times) to recover any bound origami on the filter. Be very careful not to puncture the filter during this step. Finally, add another 200uL of buffer to your sample to bring the final volume to ~400uL.

7. Spin at 100g for ~30 minutes at 4°C.

8. Remove sample for centrifuge, leaving the machine at 4°C because we’ll be using it again in a moment. The spin filter should have only a few tens of microliters of your sample left in it, just enough to cover the filter. Remove the filter and get rid of any liquid in the tube below. Now reinsert your spin filter and add 200uL of 1X buffer, then pipette up and down just over the filter (five to six times) to recover any bound origami on the filter. Be very careful not to puncture the filter during this step. Finally, add another 200uL of buffer to your sample to bring the final volume to ~400uL.

9. Spin at 100g for ~20 minutes at 4°C. This should leave you with ~70-100uL of sample retained in the spin filter. Perform the above pipetting step again to recover as many origami as possible from the spin filter.

10. Remove the spin filter, and place it upside down in a fresh tube (being careful not to dump your sample on the ground!). You can now spin your sample in a microcentrifuge for 12 minutes to collect everything.


Paul W.K. Rothemund's additional notes

A similar spin protocol appears necessary for binding DNA origami to sticky patches on silicon. Without it, the staples seem to interfere in binding. This protocol was originally taken from Rob Barish's protocol for origami/ribbon constructs. I think that some parts of the protocol may not be necessary for regular origami. Notably, I'm not sure that pipetting up and down is necessary, except perhaps at the last step to maximize recovery. Similarly the use of a 4C centrifuge seems unnecessary, the centrifugation may go much faster at room temperature. If you use a different centrifuge or temperature, calibrate the passage of buffer through the filter before you try purifying any origami to get an idea of how much buffer is retained in the filter after a spin. Fewer spins will be required if a smaller excess of staples is used. You should be able to get away with 10X fewer staples than we typically use according to my standard origami protocol, easily. That is, a 5-10X stoichiometric excess of staples over scaffold is probably fine and will result in cleaner origami faster.


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