Biomod/2011/Harvard/HarvarDNAnos:LabNotebook Sherrie

Monday (2011-06-06)
Dear Lab Notebook,
 * First day of BioMod at the Wyss Institute! This place is well furnished; I'm especially a fan of the huge sliding glass doors. Anyway, this morning we had a brainstorming session with Adam concerning box designs. We came up with three different designs. The first is a cubical box constructed with helices all going in one direction, two halves of which are apart but connected by scaffold when open and together when closed. The closing mechanism could be a tweezer design, and the lock could be opened via strand displacement. The second design is a box with helices again going in the same direction, on one face of which there is a hole closed by a mesh of staple strands binding to each other. We could design the staple strands so that the ends closing the hole all have the same sequence and can be displaced by the same key strand or cut by the same restriction enzyme. The third idea is a spherical box. We're not sure yet how to construct this sphere or how to open it, but we like the idea because it uses DNA efficiently. The box-with-helices-all-going-the-same-direction design is good because it has no seams and therefore few points of weakness, but it uses DNA rather inefficiently. Adam also suggested using 5nm gold particles as our cargo.
 * I started working on the first box design; below is a screenshot of the file in caDNAnoSQ.
 * [[Image:2011-06-06_figure1.png | 400px]]
 * When sent into CanDo, I got back a video showing thermal fluctuations of >5.5nm. That's expected, since the sides of the box not connected by scaffold are free to swing sideways. The fluctuations between helices look pretty small, so the box shouldn't leak 5nm gold particles between helices. I'm not sure if the scaffold between the two halves is long enough; when the box is in its closed state, will the scaffold bend and allow the box to close seamlessly, or will it resist bending and cause the box to have a gap? Below is a screenshot of the CanDo video.
 * [[Image:2011-06-06_figure2.png | 400px]]
 * Here's the caDNAnoSQ file I made at the end of the day.
 * [[Media:Box2.json]]

Tuesday (2011-06-07)
Dear Lab Notebook,
 * This morning, I worked on reconstructing the Andersen et al. box from a 2009 paper published in Nature. It's a box made of six roughly square sides folded together, as shown below. I tried to figure out why the Andersen box is not suitable as a robust container; there are a lot of seams, but can't we just staple them shut?
 * [[Image:2011-06-07_figure1.jpg | 400px]]
 * I soon realized, though, that there are some difficulties with stapling one side to another. Crossovers don't often match, helices are running in different directions, etc. I had a hard time figuring out where to force crossovers, especially for edges where the helices run perpendicular to each other. I'm not sure at this point if this box is worth pursuing.
 * In the afternoon, we had another brainstorming session, this time also with Tom and Wei. With Tom we talked a lot about opening and closing mechanisms for boxes. Since the distance between bases is 0.43nm in ssDNA and 0.34nm in dsDNA, he suggested using the hybridization of staple strands hanging off the lid to those hanging off the box to apply force to the lid when it's closed. He also suggested a spring mechanism to open the box. Wei informed us that 5nm gold particles aren't just 5nm in solution; they're effectively larger due to a hydrodynamic shell. So we should design our box to have an opening much larger than 5nm in diameter; Wei said at least 20nm.
 * Food for thought.

Wednesday (2011-06-08)
Dear Lab Notebook,
 * Worked all day on the box design suggested by Tom yesterday. As of now it's got one-helix thick walls, a one-helix thick bottom, and a two-helix thick lid. Here's a caDNAnoSQ screenshot of the box.
 * [[Image:2011-06-08_figure1.png | 400px]]
 * I ran into trouble when the box I initially designed had too many bases for the M13 sequence. I had to redesign the box a couple of times, making sides smaller and the box a bit shorter. It doesn't use DNA efficiently, which makes this design not so elegant. I'm not sure how to incorporate a spring opening element as Tom suggested; it seems I would need to make the box bigger to accommodate that, yet I'm constrained by the M13 sequence.
 * Later in the day we decided to embark on quest to reconstruct sphere from Han et al.'s paper in Science. It looks challenging and really interesting; we'll see how this goes!

Thursday (2011-06-09)
Dear Lab Notebook,
 * I finished a draft of the lidded box design suggested by Tom. Adam had another idea for the lid: to use stacking interactions to help hold the lid on the box. I wonder if the lid will still come off easily though? Here's the caDNAnoSQ file.
 * [[Media:Box3.json]]
 * Below is the CanDo file that came back with thermal fluctuations of the box. I'm not sure what happened with the lid; where I've attached it to the scaffold is contorted weirdly. CanDo says there are very small thermal fluctuations, though, which is great.
 * [[Image:2011-06-08_figure2.png | 400px]]
 * Then I started working on reconstructing the sphere from Han et al.'s supplemental materials. It's not as bad as I initially thought, especially since they did all the calculations for us of how many bases should be in each ring layer of the sphere, where crossovers can happen, etc. It's a matter of accurately reconstructing their design. It took me almost all day, but I finally have a scaffold of the sphere done. Adam and I took a bit of time to figure out how to construct the southern hemisphere of the sphere and connect it to the northern hemisphere, since the paper only provides instructions for designing one hemisphere. We decided a mirror image should work.

Friday (2011-06-10)
Dear Lab Notebook,
 * It's Friday! Gotta get down.
 * Continued construction of the sphere in caDNAno today, manually placing all the staple strands in the exact positions Han et al. diagrams them in Figure S10 (see below). Unfortunately, caDNAno did not make this easy. Figure S10 provides the exact nucleotide positions where staple crossovers occur; to find these positions in caDNAno, I have to use the Slide Bar in a very slow and arduous process. After five hours, I finally have the northern hemisphere all stapled. Only one more hemisphere to go...
 * [[Image:2011-06-10_figure1.jpg | 400px]]
 * In other, more uplifting news, I downloaded Maya and have started watching the short video tutorials that come with it. We plan to make a 3D model of the DNA origami sphere and write a Python script to tell us the caDNAno coordinates of any base in the 3D model. The goal is to be able to model more intuitively in 3D and then have the script translate the 3D model into caDNAno. Much more friendly than figuring the crossovers out on paper! I'm looking forward to playing around with Maya to make the model.

Monday (2011-06-13)
Dear Lab Notebook,
 * Finished the staples for the southern hemisphere, then connected the two hemispheres!! The design looks quite nice zoomed out; Evan says it's like a peacock feather. Take a look!
 * [[Image:2011-06-13_figure1.png | 400px]]
 * Before connecting the two hemispheres, I sent the file into CanDo just to see what would happen. It doesn't look like two halves of a sphere, but if you squint and tilt your head to the side...
 * [[Image:2011-06-13_figure2.png | 400px]]
 * Still doesn't look like two halves of a sphere. Oh, MIT.
 * Now I'm working on matching all my staple strands with Han et al.'s staple strands. Adam wrote a Python script to do this, and the first run was able to match 120 out of 160 staples. After I connected the two hemispheres, that number was up to 136. Adam modified the script to output which strands don't match, and from that I was able to fix some. It turns out that Han et al. didn't make all the staple strands cross over as pictured in Figure S10, which is misleading. There are also some places where bases simply don't match, e.g. there is a G in the middle of a staple strand where caDNAno says there's an A. I'm up to 140 matches right now, with the rest being these crossover or random base mismatches.
 * [[Image:2011-06-13_figure3.png | 400px]]

Tuesday (2011-06-14)
Dear Lab Notebook,
 * Continued fixing mismatched staples this morning. Got the number of staple matches up to 142, with 16 staples not matching due to seemingly random base differences (A instead of T) nowhere near crossovers. Not sure why these exist. Also weird is that 142 and 16 add to 158, which is two staples short of the 160 staples Han et al. ultimately used in their sphere. Where'd they go??
 * Here's the file with all the mismatches thus far.
 * [[Media:Sphere_mismatches.rtf]]
 * In the afternoon, I fumbled around some more with Maya and followed a tutorial to make a screwdriver! Things went pretty smoothly until I had to render using mental ray... I couldn't get the dgs_material and dielectric material of the screwdriver to render properly; they just turned up black. I spent a couple hours tweaking settings, but to no avail. That's alright, though, I learned about selecting vertices, moving stuff, duplicating stuff, scaling stuff, etc. etc. Here's what the screwdriver looks like.
 * [[Image:Screwdriver.png | 400px]]

Wednesday (2011-06-15)
Dear Lab Notebook,
 * This morning I lost my wallet and ran around calling banks and canceling cards. Fun fun fun.
 * More relevantly, I modified the staple-checking script to output all the staples that match as well as those that don't match. From this output, I color-coded the caDNAno file, making all matches black and all mismatches red. As expected, the matches and mismatches together accounted for all my staple strands. But that's still only 158; where are the other two?
 * Turns out there ARE only 158 staples. Not 160. Thanks, Han et al. All the staple strands are numbered North or South from 1 to 80, so you'd think there are 160 of them, right? Nope, not when the 14th strands don't exist at all. So the sphere I made has the right number of staples after all.
 * Okay, big picture now. Time to think about what to do with the sphere. A possible opening mechanism is getting rid of the staples holding together the two hemispheres and replacing them with a few staple strand locks that can be displaced. How do we close the hole at the top and bottom, though? A few staples crossing over the top? A cap of some sort? How big even is the hole?
 * We also imaged some long DNA origami rectangles with Ralf. We mixed staples, M13 scaffold, and folding buffer and put it all in the thermal cycler, then used the AFM to image it. Ralf and Adam also explained some AFM principles to us.
 * Continued working with Maya, this time trying to construct a DNA helix. It'd be cool if we could get helix to wind around a sphere in our Maya model.
 * Here's the sphere as of now, with staples matching Han et al.'s colored black and staples not matching theirs colored red.
 * [[Media:Sphere.json]]
 * Since there's no picture in today's entry, here's a view of what our workspace looks like:
 * [[Image:IMG_1246.JPG | 400px]]

Thursday (2011-06-16)
Dear Lab Notebook,
 * This morning I worked on designing locks along the equator of the sphere. I used Nick's excel spreadsheet to find all the bases pointing outward, which is where I can put the locks.
 * In the afternoon, the team met with Peng Yin, William Shih, and other lab members to discuss our progress and how to proceed. We talked a lot about ways to solubilize the cargo inside the box. Shih suggested that we use disulfide bonds, which are stable at high temperatures and can be cleaved with the addition of DTT to solution, to connect the cargo to the inside of the box. We can also use strand displacement; it's possible that single strands can diffuse inside the sphere (though slowly) through the top and bottom holes. Azobenzene is another option, as is restriction enzymes. Not sure if restriction enzymes will diffuse through ~4nm holes though.
 * We also talked about another interesting design for a box that we can explore later: a torus! The nice thing about a torus is that it's a totally closed space. This idea applied to the sphere we have is just to run DNA rings through the center of our sphere to close it off. Or, maybe we can make a cylinder of DNA rings, then bend them to form a torus, then cut the torus at one ring to release cargo.
 * [[Image:2011-06-16_figure1.JPG | 400px]]
 * List of things we want to construct:
 * Normal sphere
 * Sphere with disulfide on equator and cargo handle staples
 * Sphere with strand displacement cargo handle staples, disulfide on equator
 * Sphere with strand displacement equator staples, disulfide handle staples
 * Sphere with restriction enzyme staples
 * Sphere with azobenzene staples
 * Cubical box

Friday (2011-06-17)
Dearest Lab Notebook,
 * Created a spreadsheet with "equator" staple sequences for a disulfide sphere and a strand displacement sphere.
 * [[Media:Sphere_staples.xlsx]]
 * To do this, I used Nick's spreadsheet to find where staple strands along the sphere's equator face outward on the northern and southern hemispheres, then where these outward strands line up. I also designed tweezers with staple strand extensions, a lock strand, and a key strand complementary to the lock strand. The current sequence I have is taken from the Liang paper, so I assume it will work better than something I randomly generate (but maybe not). I did, however, have to come up with my own addition to the lock and key strands, as the Liang paper doesn't use strand displacement to open the tweezers. Here are the NUPACK results of the lock and key, respectively.
 * [[Image:NUPACK1_lock.png | 400px]]
 * [[Image:NUPACK1_key.png | 400px]]
 * The staple extensions are universal; that is, the same staple extensions will be added to the ends of all staples on the equator so they can be locked with the same lock and opened with the same key. It'd be interesting to explore different tweezer sequences and see which ones hold the sphere closed best, which ones are easiest to open, etc. It'd also be neat to vary the number of locks holding the sphere closed; is 2 enough? How long will it take 9 to open?

Monday (2011-06-20)
Ahoy there Lab Notebook,
 * This morning we talked to Tom about the forces that hybridization of DNA strands can provide on an origami box. According to this paper, the force exerted by a DNA lock in the shear geometry (see below) is ~50 pN. I'm not sure how big this is for a DNA origami box, but according to Tom we can calculate how much 50 pN will cause a DNA lid to bend using the same equations engineers use for buildings, which is pretty cool.
 * [[Image:2011-06-20_figure2.jpg]]
 * Tom also brought up a potential problem for assembling the lid: if one lock hybridizes first, it may separate the other sides of the lid from the box such that the other locks can't lock. Then all our boxes will be open, because it's almost guaranteed that one lock hybridizes before the others. One potential solution is to design the lock opposite the lid hinge such that it hybridizes first; another solution is to add the opposite lock strand first, then the side lock strands.
 * [[Image:2011-06-20_figure1.jpg | 400px]]
 * In the afternoon I made a schematic of the strand displacement/tweezer method for opening the sphere.
 * [[Image:2011-06-20_figure3.jpg | 400px]]

Tuesday (2011-06-21)
Dear Lab Notebook,
 * We started experiments today! In the morning we diluted the DNA we're using for disulfide cross-linking and gold nanoparticle conjugation.
 * In the afternoon, Steve showed us how to use the dynamic light scattering (DLS) machine to figure out the diameter of gold nanoparticles. The stock of 5nm gold particles that Adam has showed some aggregation of nanoparticles (it's been in a fridge for a while) with hydrodynamic diameters of 50nm and 5um. Here's the data file the machine output:
 * [[Media:Gold_DLS.txt]]
 * Later, Evan and I continued diluting the DNA (oh yay stoichiometry) and mixed an M13 ultramer with an increasing number of oligonucleotides to create strands of DNA off which hang oligonucleotides with tails. The goal is to eventually attach gold nanoparticles to this strand and get a "necklace" of sorts.

Wednesday (2011-06-22)
Dear Lab Notebook,
 * This afternoon we made our own 5nm, 15nm, and larger gold nanoparticles with Steve. You just heat gold citrate and add stabilizer and reducing agent (citrate and tannic acid/citrate depending on the size of the particles desired); pretty simple! They came out pretty well, we think. They also came out pretty, period.
 * [[Image:IMG_1270.JPG | 400px]]
 * We then used dynamic light scattering to characterize the nanoparticles, and you can see below the hydrodynamic diameters of the nanoparticles.
 * [[Image:DLS_gold5nm.png | 400px]]
 * [[Image:DLS_gold5nm(2).png | 400px]]
 * [[Image:DLS_gold15nm.png | 400px]]
 * We also ran a gel with our ultramer and increasing numbers of handles, but the gel broke the first time so we had to do it again. The second try came out well, though; you can see below the shift in bands as the ultramer has more handles connected to it. We didn't run it for enough time so the bands haven't quite separated, so we'll do it again.
 * [[Image:GEL20.JPG | 400px]]
 * Also. Nick is a mad scientist.
 * [[Image:IMG_1272.JPG | 400px]]

Thursday (2011-06-23)
Dear Lab Notebook,
 * Spent the morning concentrating gold nanoparticles and working intermittently on a powerpoint presentation to be given at the Yin lab meeting. Finished the powerpoint in the afternoon and presented first at lab meeting. Some useful feedback:
 * Thiolated nanoparticle strand needs to be distinct from the disulfide
 * Strand (to allow conjugation to gold without breaking the disulfide - gold itself can competitively reduce disulfides)
 * Box foldings with AuNP cargo should be 2-step not 1-step
 * Linking of 5' S-S strand directly to gold particle works with high efficiency. We have one of these for the 15-mer, but it is short, so gel separation won't be good. Still, we can try it
 * Look into conditions necessary for restriction enzyme activity
 * Caged DNA instead of azobenzene: impractical this summer since we have the azobenzene strand already
 * Use type-II restriction enzyme which is an "offset cutter" to cut close to origami while keeping cut site a reasonable distance away from the origami

Friday (2011-06-24)
Dear Lab Notebook,
 * This morning we reran an ultramer-handle band shift gel and, now that we know the handles are binding to the ultramer, also ran a gel containing lanes with the ultramer and all the handles. The latter gel is for purification of ultramers containing all the handles.
 * Things to test for strand displacement opening mechanism:
 * Number of locks needed to close sphere (find minimum)
 * Amount of time it takes to open sphere with N locks
 * Tweezer sequences (find type [A/T? C/G?] with highest yield for opening/closing)
 * Tweezer lengths (what range to test?)

Monday (2011-06-27)
Dear Lab Notebook,
 * Today we tried a gel extraction of the gold nanoparticles conjugated to DNA based on instructions from Steve. It didn't seem to work, though, as the gold nanoparticles migrated extremely slowly and not in distinct bands. I wonder what went wrong... We're gonna wait for Wednesday (when Steve gets back) to try again.
 * [[Image:IMG_1276.JPG | 400px]]
 * I also finished up revising staples for the strand displacement opening mechanism, and now have a new document of sphere staples.
 * [[Media:Sphere_staples_updated.xlsx]]
 * In the afternoon, Nick and I figured out where we wanted to place our handles for loading cargo. Then I spent an annoyingly large amount of time sorting all the sphere staples into groups: those that are "Han" staples that are neither lock nor handle, those that are "my" staples, those that are "Han" lock staples, and those that are "Han" handle staples. Tomorrow I can start pooling the strands and hopefully folding the sphere!
 * [[Media:Sphere_wells.xlsx]]

Tuesday (2011-06-28)
Dear Lab Notebook,
 * This morning I pooled the sphere staple strands according to the document I created yesterday. In total there are now 28 pools: one with 109 staples that match Han's and aren't part of the lock or handle mechanisms, one with 16 staples that don't match Han's, 25 with locks, and four with handles.
 * Then, in the afternoon, I made four reaction solutions: one using buffer with 12.5mM MgCl2, one using buffer with 12.5 MgCl2 and gold nanoparticles, one using buffer with 16mM MgCl2, and one using buffer with 16mM MgCl2 and gold nanoparticles. With the gold nanoparticles, we're curious just to see if the gold will be folded inside the sphere by chance, and what that yield is. The mixtures are currently in the thermocycler, following the cycle given by the experimental methods from the Han paper. In 12 hours we'll see if we have spheres!

Miércoles (2011-06-29)
Querido laboratorio portátil,
 * Esta mañana, preparé un gel con las esferas. También les ayudé a Evan y Nick preparar geles para sus experimentos. Terminé las secuencias para las grapas con manejas; puedes ver la hoja de cálculo debajo.
 * [[Media:Sphere_staples2.xlsx]]
 * Usamos el AFM para obtener un imagen de las esferas. No es ideal para 3D origami, sin embargo podemos ver algunas formas circulares que esperamos que sean las esferas. Aquí está un imagen de "las esferas":
 * [[Image:Sphere_1.jpg | 400px]]
 * Esta tarde, purifiqué las esferas con el gel; corté la sección roja en el gel (debajo), la trituré, y la filtré. Puedes observar en los lanes con 8mM MgCl2 buffer las rayas del M13 scaffold; no hay origami. En la ponencia de Han et al., se usó 16mM MgCl2 buffer. En los lanes con este buffer, puedes observar origami. No sé por qué hay muchas rayas en el lane de M13 scaffold... debe existir sólo una.
 * [[Image:2011-06-29_figure1.jpg | 400px]]
 * Usé el AFM para obtener un imagen de estas esferas purificadas, y aquí está. No estamos seguro de que sean esferas o no.
 * [[Image:Sphere_purified.jpg | 400px]]

Thursday (2011-06-30)
Dear Lab Notebook,
 * Did TEM imaging this morning with Wei. We're not sure if we saw spheres or just water droplets, though... The staining wasn't great so it was hard to tell. We did see gold nanoparticles, though; they're black under TEM. See for yourself.
 * [[Image:Sphere1_TEM.jpg | 400px]]
 * Above there's a circular shape that is around the right size to be a spherical origami. Or just a drop of water.
 * [[Image:Sphere2_TEM.jpg | 400px]]
 * Here you can see the gold nanoparticles, around 5nm as they should be. The lighter circles look promising, but are too small to be our origami; they're ~20nm in diameter while our origami should be ~40nm.
 * If we indeed did not see any spheres in the purified samples, possible explanations include:
 * Purification did not work
 * Staining was sub-optimal
 * Spheres did not fold (we had 16 imperfectly matched staples, though this shouldn't prevent the whole structure from forming)
 * The band we took from the gel did not contain spheres
 * Things to try:
 * Image unpurified spheres
 * Stain unpurified and purified spheres again with Wei
 * (If we see spheres in the unpurified sample but not the purified) Move our way up the gel, purifying different sections of the smear
 * In the afternoon, we had a meeting with William Shih and Peng Yin, who reminded us that gold nanoparticles cleave disulfide bonds, meaning we probably can't use disulfide bonds as an opening/closing and solubilization mechanism. Instead they suggested a photo-cleavable linker, which we had previously considered but not pursued. I updated the inventory of sphere staples and designed new photo-cleavable equator staples and handles.

Friday (2011-07-01)
Dearest Lab Notebook,
 * Per Wei's suggestion, this morning I used the nanodrop to measure the concentration of origami in the purified and unpurified sphere samples. According to Wei, the A260 column should read around 0.15 or 0.2. This was indeed the case.
 * {| class="wikitable" border="1"

! Sample ! A260 Reading
 * Unpurified sphere
 * align="right" | 16.851
 * Purified sphere gel position 1
 * align="right" | 0.145
 * Purified sphere + AuNP gel position 1
 * align="right" | 0.135
 * Purified sphere + AuNP gel position 2
 * align="right" | 0.199
 * Purified sphere + AuNP gel position 3
 * align="right" | 0.151
 * Purified sphere + AuNP gel position 4
 * align="right" | 0.151
 * Purified sphere + AuNP gel position 5
 * align="right" | 0.170
 * Purified sphere + AuNP gel position 6
 * align="right" | 0.131
 * Purified sphere + AuNP gel position 7
 * align="right" | 0.160
 * }
 * In the afternoon, we ran a gel of the boxwithlid. I also re-ran a gel with the sphere to confirm what we got last time. Wei helped us prep samples for the TEM, and he'll image them for us later today (thanks Wei!) since the TEM is booked until 6ish.
 * Also changed the color scheme of the wiki. No more crimson!
 * Purified sphere + AuNP gel position 6
 * align="right" | 0.131
 * Purified sphere + AuNP gel position 7
 * align="right" | 0.160
 * }
 * In the afternoon, we ran a gel of the boxwithlid. I also re-ran a gel with the sphere to confirm what we got last time. Wei helped us prep samples for the TEM, and he'll image them for us later today (thanks Wei!) since the TEM is booked until 6ish.
 * Also changed the color scheme of the wiki. No more crimson!
 * In the afternoon, we ran a gel of the boxwithlid. I also re-ran a gel with the sphere to confirm what we got last time. Wei helped us prep samples for the TEM, and he'll image them for us later today (thanks Wei!) since the TEM is booked until 6ish.
 * Also changed the color scheme of the wiki. No more crimson!

Tuesday (2011-07-05)
Dear Lab Notebook,
 * After we left on Friday, Wei imaged the unpurified and purified sphere samples again and found no spheres. Perhaps the folding reaction was not ideal for the formation of spheres; I used the protocol Adam gave me, which asks for 5x staple excess. Han's protocol calls for 10x staple excess. We thought the M13 scaffold was degraded and bad because of the multiple bands on my first gel, but Adam made some origami with it this weekend, so it should be fine.
 * More importantly, I also realized that, in the protocol for basic origami folding, by "add 5 uL buffer," "buffer" refers to 10x folding buffer instead of 1x folding buffer. 5 uL in 50 uL is a 10x dilution, after all... welp, that's probably why there were no spheres in the last batch. I was off by a factor of 10. Muy bien Sherrie.
 * (Still, strange though that the lanes from the last gel with 1.6 mM MgCl2 had a smear, while those with 8 mM MgCl2 did not...)
 * So I remade some 10x Han Buffer 2, added scaffold and the necessary 10x excess staples, and put it all in the thermocycler. I also folded both spherical origami and spheres without equator strands; the latter should be easier to distinguish on the AFM and TEM. Hopefully this time we'll see spheres!
 * In other news, a new logo is up. Me gusta.
 * In other other news, we're fighting about where the "Literature" link belongs on the front page. This is what happens when you put four anal people together.

Wednesday (2011-07-06)
Dear Lab Notebook,
 * Round 2 of sphere ordering is done! Instead of disulfide linkers, I switched our handles and equator staples so they now have photo-cleavable (PC) spacers. Originally I replaced every disulfide link with a PC spacer, but it turns out that PC spacers are maaaaad expensive. So we came up with the idea to alter our lock strand to have the PC spacer; that way, we only need to order one (instead of 18) PC strands for the opening mechanism. The four handles all have a PC spacer, too.
 * In the afternoon we ran a gel of the sphere samples.
 * [[Image:2011-07-06_Spheregel.jpg | 400px]]
 * We also used the AFM and TEM to image the closed and open spheres, and got some really exciting results! The AFM images look really promising.
 * Unpurified closed spheres:
 * [[Image:2011-07-06_Sphereunpurifiedclosed_AFM1.jpg | 400px]]
 * [[Image:2011-07-06_Sphereunpurifiedclosed_AFM2.jpg | 400px]]
 * Unpurified open spheres:
 * [[Image:2011-07-06_Sphereunpurifiedopen_AFM1.jpg | 400px]]
 * [[Image:2011-07-06_Sphereunpurifiedopen_AFM2.jpg | 400px]]
 * One of the coolest things is that you can see dimers in the AFM image of open spheres. Perhaps those are the two sphere hemispheres.
 * The TEM images are more ambiguous. Spherical shapes could be water droplets, dirt, etc. But still promising.
 * Unpurified closed spheres:
 * [[Image:2011-07-06_Sphereunpurifiedclosed_TEM.jpg | 400px]]
 * Unpurified open spheres:
 * [[Image:2011-07-06_Sphereunpurifiedopen_TEM.jpg | 400px]]

Thursday (2011-07-07)
Dear Lab Notebook,
 * Gel purified spheres this morning for imaging. I cut the gel in places indicated in the diagram from yesterday. Also prepped samples for TEM imaging. Unfortunately, the success rate of prepping was rather low... those tiny copper grids are hard to work with.
 * Tried AFM on the purified closed sample, but we didn't see much. The TEM was booked this afternoon, so we'll do that tomorrow.
 * I also prepared 48-hour reactions for folding the spheres, one closed and one open. Perhaps folding for a longer time will result in better-folded structures that we can see under AFM and TEM.

Monday (2011-07-11)
Dear Lab Notebook,
 * I tried to spin column purify 20 uL of the 48-hour spheres this morning, but ended up with next to no product. Measurements with the nanodrop resulted in these measurements:
 * {| class="wikitable" border="1"

! Sample ! UV-Vis 260 Reading
 * Purified closed sphere 1
 * align="right" | 0.005
 * Purified open sphere 1
 * align="right" | 0.006
 * Purified closed sphere 2
 * align="right" | 0.013
 * Purified open sphere 2
 * align="right" | 0.014
 * }
 * In other words, there's basically no origami in the purification product.
 * Later, I ran a gel of the closed and open spheres and got this:
 * [[Image:2011-07-11_spheregel.jpg | 400px]]
 * Where'd the closed spheres go?? Did I forget scaffold or something...
 * In the afternoon I redid 12-hour reactions. Tomorrow I want to use AFM to image various open sphere bands from the first sphere gel, and run gels on the new 12-hour sphere reactions.
 * Later, I ran a gel of the closed and open spheres and got this:
 * [[Image:2011-07-11_spheregel.jpg | 400px]]
 * Where'd the closed spheres go?? Did I forget scaffold or something...
 * In the afternoon I redid 12-hour reactions. Tomorrow I want to use AFM to image various open sphere bands from the first sphere gel, and run gels on the new 12-hour sphere reactions.

 