Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Protocols
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(→How to normalize SPEX Data in MATLAB) 
(→How to normalize SPEX Data in MATLAB) 

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*The data is normalized such that the start of the reaction is at 1, and the completion level is at 0, by translating the completion level down, and then scaling the data.  *The data is normalized such that the start of the reaction is at 1, and the completion level is at 0, by translating the completion level down, and then scaling the data.  
:<syntaxhighlight lang="matlab">time1 = data(1, start:finish);</syntaxhighlight>  :<syntaxhighlight lang="matlab">time1 = data(1, start:finish);</syntaxhighlight>  
  time2 = data(3, start:finish);  +  :<syntaxhighlight lang="matlab">time2 = data(3, start:finish);</syntaxhighlight> 
  time3 = data(5, start:finish);  +  :<syntaxhighlight lang="matlab">time3 = data(5, start:finish);</syntaxhighlight> 
  time4 = data(7, start:finish);  +  :<syntaxhighlight lang="matlab">time4 = data(7, start:finish);</syntaxhighlight> 
  data1 = data(2, start:finish);  +  :<syntaxhighlight lang="matlab">data1 = data(2, start:finish);</syntaxhighlight> 
  data2 = data(4, start:finish);  +  :<syntaxhighlight lang="matlab">data2 = data(4, start:finish);</syntaxhighlight> 
  data3 = data(6, start:finish);  +  :<syntaxhighlight lang="matlab">data3 = data(6, start:finish);</syntaxhighlight> 
  data4 = data(8, start:finish);  +  :<syntaxhighlight lang="matlab">data4 = data(8, start:finish);</syntaxhighlight> 
  time1 = time1  time1(1);  +  :<syntaxhighlight lang="matlab">time1 = time1  time1(1);</syntaxhighlight> 
  time2 = time2  time2(1);  +  :<syntaxhighlight lang="matlab">time2 = time2  time2(1);</syntaxhighlight> 
  time3 = time3  time3(1);  +  :<syntaxhighlight lang="matlab">time3 = time3  time3(1);</syntaxhighlight> 
  time4 = time4  time4(1);  +  :<syntaxhighlight lang="matlab">time4 = time4  time4(1);</syntaxhighlight> 
  ndata1 = data1  data1(finish  start + 1);  +  :<syntaxhighlight lang="matlab">ndata1 = data1  data1(finish  start + 1);</syntaxhighlight> 
  ndata2 = data2  data2(finish  start + 1);  +  :<syntaxhighlight lang="matlab">ndata2 = data2  data2(finish  start + 1);</syntaxhighlight> 
  ndata3 = data3  data3(finish  start + 1);  +  :<syntaxhighlight lang="matlab">ndata3 = data3  data3(finish  start + 1);</syntaxhighlight> 
  ndata4 = data4  data2(finish  start + 1);  +  :<syntaxhighlight lang="matlab">ndata4 = data4  data2(finish  start + 1);</syntaxhighlight> 
  ndata1 = ndata1/ndata1(1);  +  :<syntaxhighlight lang="matlab">ndata1 = ndata1/ndata1(1);</syntaxhighlight> 
  ndata2 = ndata2/ndata2(1);  +  :<syntaxhighlight lang="matlab">ndata2 = ndata2/ndata2(1);</syntaxhighlight> 
  ndata3 = ndata3/ndata3(1);  +  :<syntaxhighlight lang="matlab">ndata3 = ndata3/ndata3(1);</syntaxhighlight> 
  ndata4 = ndata4/ndata4(1);  +  :<syntaxhighlight lang="matlab">ndata4 = ndata4/ndata4(1);</syntaxhighlight> 
  plot(time1, ndata1, time2, ndata2, time3, ndata3, time4, ndata4);</syntaxhighlight>  +  :<syntaxhighlight lang="matlab">plot(time1, ndata1, time2, ndata2, time3, ndata3, time4, ndata4);</syntaxhighlight> 
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Revision as of 19:33, 12 October 2011
Tuesday, April 21, 2015

Protocols
Things that need to be written
Gel Purification
For 300ul sample, add 50ul BB_ND dye 170ul of sample will be loaded per lane. Samples will take 2 lanes, and one blank lane should be placed between different samples.
 Feel it with fingers/ Watch out for the water flow
Lulu’s recipe for 40ml of 12% ND gel which works for one gel  12ml 40% Acrylamide  4ml 10x TAE/Mg++  Fill up to 40ul MQ water (add 24 ml)  240ul 10% APS, slow mix  24ul TEMED, slow mix APS should be no more than 10 days old
SPEX protocol
Bottom big on/off button is for the machine. It is always on. Top small one is for the lamp. If there’s somebody who will use the SPEX tomorrow, then do not turn off the lamp after the experiment.
Default: Click OK
Click the third square icon called “Visual setup” Click the middle square Temperature control icon: Turn on and Set to 25 degreeC Choose T bath (internal). NOT Probe (external).
Be careful! Hold only top or bottom. DW (10 times) 70% ethanol (twice) DW (5 times) 70% ethanol (once) While you wash, you can touch the side. Hold it in a stable way. Let it dry (upside down)
High voltage: HV on, S 950, T 950 Bandpass : Set 2 for everything
Open a file called “ROX_CWA” from e:\SPEX DATA\biomod Optional mode: kinetics, Integration time: 10s Define: Repeat acquisition: 1 min (minimum) for total of 180 min (3 hours)
Range of the real data should not exceed 1 million (1000,000) Number we see is S/R will be (100 million)
When the status is “waiting”, click “stop”. Don’t click anything else. Add and mix. After you put the cuvettes inside the box, click “no”.
When the status is “waiting”, click “stop”. Click “yes” Save data in two forms (DWA datafile, multi~ test) Check if the data is properly saved: Check the last data.
Origami Purification
We suggest using the Millipore Microcon YM100 (blue) spin filters. ( http://www.millipore.com/catalogue/item/42413 ). 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 substrateladen samples. 1. Mix 100uL of your sample with 300uL of 1X buffer of your choice. 2. Vortex mixture and pipette it into a YM100 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 ~70100uL 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.
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 510X stoichiometric excess of staples over scaffold is probably fine and will result in cleaner origami faster.
How to Open SPEX Data in MATLAB
How to normalize SPEX Data in MATLAB
