Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Protocols/Origami Purification - Revision history

YAE LIM Lee at 07:15, 3 November 2011

YAE LIM Lee — Thu, 03 Nov 2011 07:15:18 GMT

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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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YAE LIM Lee at 07:15, 3 November 2011

YAE LIM Lee — Thu, 03 Nov 2011 07:15:00 GMT

←Older revision		Revision as of 07:15, 3 November 2011
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	== Origami Purification ==		== Origami Purification ==

YAE LIM Lee at 03:53, 3 November 2011

YAE LIM Lee — Thu, 03 Nov 2011 03:53:01 GMT

←Older revision	Revision as of 03:53, 3 November 2011
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	+	== Origami Purification ==
	+
	+	====Provided by Nadine Dabby and Sungwook Woo====
	+
	+	We suggest using the [http://www.millipore.com/catalogue/item/42413\| 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.

YAE LIM Lee: Removing all content from page

YAE LIM Lee — Thu, 03 Nov 2011 03:51:53 GMT

Removing all content from page

←Older revision		Revision as of 03:51, 3 November 2011
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-	~~== SPEX==~~
-
-	~~1. Turn on the lamp and wait for 30 min for the lamp to warm up.~~
-
-	~~2. Launch the software: Instrument control center~~
-
-	~~3. Temperature set up~~
-	~~Click the third square icon “Visual setup”~~
-	~~Temperature control icon: Turn on and Set to 25 degreeC~~
-	~~Choose T bath (internal).~~
-
-	~~4. Wash the cuvettes.~~
-	~~Distilled Water (DW) (10 times) 70% ethanol (twice) DW (5 times) 70% ethanol (once)~~
-	~~Air dry the cuvettes for 1 h.~~
-
-	~~5. Clean the cuvettes using lens paper.~~
-
-	~~6. Click second square icon “real time display”.~~
-	~~High voltage: HV on, S 950, T 950~~
-	~~Bandpass : 2~~
-
-	~~7. Click the last icon “run CWA”.~~
-	~~Open the file “ROX_CWA”~~
-	~~Optional mode: kinetics, Integration time: 10s~~
-	~~Define: Repeat acquisition: 1 min (minimum) for a total of ~ min, depending on how long you would like to run the experiment.~~
-
-	~~8. Mixing: Mix the sample with 1X TE/Mg++ buffer inside cuvettes by pipetting for 30 times.~~
-
-	~~9. Start acquisition. Run sample.~~
-	~~The data should not exceed 1 million (1000,000).~~
-
-	~~10. To add more samples (for example, walker triggers) in the middle of SPEX experiment~~
-	~~When the status is “waiting”, click “stop”.~~
-	~~Add samples and mix. Replace cuvettes into the SPEX machine.~~
-
-	~~11. Save the data~~
-	~~When the status is “waiting”, click “stop”.~~
-	~~Click “yes”~~
-	~~Save data in two forms (DWA datafile, txt file)~~
-	~~Check if the data is properly saved.~~
-	*If you want to see the reference signals, go to options: view option and display raw R.

YAE LIM Lee: New page: == SPEX== 1. Turn on the lamp and wait for 30 min for the lamp to warm up. 2. Launch the software: Instrument control center 3. Temperature set up Click the third square icon “...

YAE LIM Lee — Thu, 03 Nov 2011 03:51:25 GMT

New page: == SPEX== 1. Turn on the lamp and wait for 30 min for the lamp to warm up. 2. Launch the software: Instrument control center 3. Temperature set up Click the third square icon “...

New page

== SPEX==

1. Turn on the lamp and wait for 30 min for the lamp to warm up.

2. Launch the software: Instrument control center

3. Temperature set up
Click the third square icon “Visual setup”
Temperature control icon: Turn on and Set to 25 degreeC
Choose T bath (internal).

4. Wash the cuvettes.
Distilled Water (DW) (10 times) 70% ethanol (twice) DW (5 times) 70% ethanol (once)
Air dry the cuvettes for 1 h.

5. Clean the cuvettes using lens paper.

6. Click second square icon “real time display”.
High voltage: HV on, S 950, T 950
Bandpass : 2

7. Click the last icon “run CWA”.
Open the file “ROX_CWA”
Optional mode: kinetics, Integration time: 10s
Define: Repeat acquisition: 1 min (minimum) for a total of ~ min, depending on how long you would like to run the experiment.

8. Mixing: Mix the sample with 1X TE/Mg++ buffer inside cuvettes by pipetting for 30 times.

9. Start acquisition. Run sample.
The data should not exceed 1 million (1000,000).

10. To add more samples (for example, walker triggers) in the middle of SPEX experiment
When the status is “waiting”, click “stop”.
Add samples and mix. Replace cuvettes into the SPEX machine.

11. Save the data
When the status is “waiting”, click “stop”.
Click “yes”
Save data in two forms (DWA datafile, txt file)
Check if the data is properly saved.
*If you want to see the reference signals, go to options: view option and display raw R.