Here are the results of the reaction that I posted earlier today. In lane 1 is a 1kb DNA ladder. The second visible band from the bottom is 1kb in length. The next band is 1.5kb. Lanes 2-6 are each 10ul (mixed with 2ul of 6x loading buffer) from their respective PCR reaction tubes (all the same reaction). Lane 2 clearly worked the best, while lane 3 apparently contained a failed reaction. The other lanes (4-6) worked well enough in this test run. And as you can see, since the band travel distance is right between the 1kb and 1.5kb mark, that puts these fragments at right around 1.1kb (primers annealed at ~980 and 2008 on the template strand). More tests will need to be conducted in the future, but the preliminary results appear quite promising.
Note: It should be noted that this gel does not display a PCR reaction after cleanup. I went straight from the thermal cycler to the gel. The faint bands at the bottom of the image are the primers used in the PCR reaction.
This reaction is based on the reaction for making the 1.1kb anchor used in the unzipping construct, which I haven’t discussed in this space, will in the future, and some background can be found on OpenWetWare.
This particular reaction makes a 1.1kb dsDNA product that is labeled on one end with a digoxygenin molecule and has no label on the other end. The reaction can be found here.
In most of these samples there is one seedling that shed the seed coat, and in few that seedling is floating at the water surface. It also seems that the branching in the second DD water sample (the Virginia Gold species) has been shed on one of the seedlings (perhaps the one that shed the seed coat). There is a stringy thing floating in the background, which I’m not sure is visible in the image but definitely there in the sample.
Note: There is some wordpress gallery error that won’t display the second and third picture captions. They are: 2. Dark V in Tap Water. 3. Dark V in DI Water
These images are from the presoaked tobacco seeds. I soaked them in their respective buffers (deuterium depleted water (DD water), tap water, and deionized water (DI water)), and then stored them in the fridge (4C) for 5 days in case there is any drastic chemical exchange between the seed and the water (which would change the buffer). It appears that the deuterium depleted water has started sprouting first in both cases, which is speculative but noteworthy. Also the branching that I noted in the last post appears again here, but only in the DD water samples, which is interesting. I wonder what that could be.
And as an aside I had to find a way to display syntax in the blog. First I tried Syntax Highlighter Compress, but that didn’t work and so then I learned that WordPress plugins are called using shortcode syntax which led me to Show Shortcode. Using this plugin is just the shortcode:
[showshortcode][/showshortcode]
And you place your shortcode in between the tags.
Note: The use of shortcodes is very much like coding in HTML except in some cases (like the Mindmeister plugin) no closing tag is needed.
Mindmeister and WordPress apparently do not work well together. I spent a good hour trying to figure out how iframes work in WordPress and it turns out they don’t so I had to install a plugin. I used Embed Iframe and it works, but it turns out that (well at least for me) Mindmeister wants to stick a big panel in front of the mindmap blocking at least 50% of the map. Booo! There is a mindmeister plugin, but I don’t know if I want to play with it right now. Maybe later, and then I’ll tell you all about it.
Back on track, if the above mindmap embed doesn’t work for you then the link is here to see it in full page glory.
Update: I found out that there was a plugin for Mindmeister and replaced the previous iframe coding with the new plugin. The process of discovery can be found here.
Several months ago, a colleague (Andy Maloney) found out about a movement to make an open source PCR machine (thermal cycler) and signed the lab up immediately to receive one of the first ones off the assembly line. It is produced by a company known as OpenPCR and was made by two amazing gentlemen (Tito Jankowski and Josh Perfetto) with support from a great community.
The machine itself costs $599 which is considerably cheaper then the cheapest options (a quich search revealed a low of at least $1500 new). It is a Do-It-Yourself (sort of) build with very detailed step by step instructions, which helps keep the cost low. Also the implementation of the Arduino chip also helps in the cost department but is also why OpenPCR can be completely open sourced.
pieces for the start of the build
outside of lid
completed lid
lid alternate angle
pcr core
lid, core, display assembly
lid, core, display side view
electronics and casing
pic before final assembly
wires strewn about
wires organized
build complete
pcr top view
top/back view
credits!
Above is an image gallery of the build. I took a picture after I completed each section of the build instructions, which thankfully were meticulously detailed and very easy to follow. I liken it to building lego playsets in my childhood. Along with the detailed instructions, all the parts were contained in numbered and lettered boxes and bags (respectively) which would be called in the instructions. This made finding parts extremely easy and quick.
Once the build was complete the machine needed a quick power test and connection to a computer or laptop. The PCR reaction programs are set via this connection through a very elegant and simple user interface. If you are constantly changing programs the thermal cycler will need to remain tethered to the computer, but if you use the same cycle over and over you can unplug and the program will run when the machine is turned on again.
I ran a series of tests to determine the capabilities and consistency of the machine. They provide a simple program appropriately named “Simple Experiment” that will do two PCR cycles (90C, 55C, 72C) and hold at 20C to test the machine. There is even a heated lid which you can set the temperature of, a very nice feature. The software gives you great flexibility when designing a reaction program. You can adjust temperatures, time, cycles, add steps, remove steps, change the lid temp, and set the final hold temp (but from my own studies and from email communiques the machine can’t handle anything below 10C).
After running the “Simple Experiment” I discovered that there were some errors with the cool down and hold feature. In some cases the hold temp works just fine (unless the temp is set too low), in others the hold feature crashes and the machine will just naturally cool instead of being temperature controlled. In one (and only one) test, the reaction program restarted when the hold stage was reached and then initiated when that program ended.
Other than this one issue, I’ve found that OpenPCR is very consistent temperature and time wise. I ran a reaction that would cycle between 94C, 55C, and 72C ten times (for 30 sec each step), then go to 37C and hold there for 10 min, then hold at 20C. It seems to never accurately reach temperature (it goes to 90C but that may be an upper limit, then 55C, then 70C), but it is very close and perhaps close enough. The main problem like I said is the hold programming which in this data set does not work. After the 37C for 10 min the program ended and the heating block (where the PCR tubes sit) cooled via air conduction instead of temperature cooling. It never reached 20C.
Bottom line though is that OpenPCR works very well and the support staff (Tito and Josh) are very helping, knowledgeable, and open to improving the product. In fact I’m scheduled to meet with them via phone tomorrow to share with them all of my findings in gory details. I’ll let you know how it goes! Until then…
I am growing 2 different species of tobacco seed (Virginia Gold #1 and Dark Virginia purchased from The Tobacco Seed Company, as an aside I find it strange that we bought seeds from a company in England that gets their seeds from the United States) in different water buffers: regular tap water, 18MΩ deionized water (DI water), and deuterium depleted water (DD water).
I place 3 seeds of each species in a cuvette (I actually have no clue what company these are from because they are from a former student in the lab, but USA Scientific has a comparable type) and add one type of water. So there are a total of 6 cuvettes (3 for each species).
Seeds in water in cuvettes.
The experiment has way more to consider than I initially suspected which presents some interesting challenges. On top of that I don’t know all that much (right now) about how deuterium interacts with the environment and the seeds, and I don’t know the biochemistry of seed growth in general. Because of this I started a second set of experiments that are identical except that the seeds were presoaked in their respective buffers in the refrigerator in case the initial stages of growth dramatically changed the water solution.
Finally I’m in the process of figuring out how to accurately record growth rates and right now I’m using very primitive macro photography (my camera phone and a big magnifying lens), which will develop into more advanced macro photography (Dr. Koch’s personal DSLR with 10x magnification lens) and hopefully eventually evolve into the microscope and camera system. Here is my current photography setup:
Cuvette photography system.
I have a 2in lens with a focal length of about 3.5in setup on an adjustable post (which is mounted on a rail). I place cuvettes on a cylindrical lens holder (the clampy thing in the back) and adjust the lens height and distance to get the best picture. Most parts are opto-mechanics purchased from Thor Labs.
Hydrogen has several isotopes and one of them, deuterium, exists quite naturally in water to form . In previous experiments and severalpapers by Gilbert Lewis, it has been found that life is hindered in the presence of . While this may be true, my PI Steve Koch wondered if life had found a use for it because naturally occurring water has about a 17mM (millimolar) concentration of deuterium.
To put that number into perspective, when I do a typical polymerase chain reaction of DNA I add 10mM of each base of DNA (which is less than the amount of naturally occurring deuterium) to create millions of copies of a DNA template from an amount that is 1000x less then what the reaction yields. In fact most chemicals in most of my buffers on the order of the amount of naturally occurring deuterium.
So you can see it isn’t a stretch to think that nature has found a use for since it is quite abundant and life has been constantly evolving for billions of years. I want to test this hypothesis in a variety of different organisms:
Tobacco Seeds – to act as a foil to Lewis’ experiments in which he grew tobacco seeds in pure .
Mustard Seeds – from what I’m told mustard seeds are the powerhouse of the botanical genetics world much like Drosophila and S. cerevisiae are in their respective genetic fields.
Escherichia coli – another molecular biological powerhouse that is very easy to grow and may be easy to see results with. We just got the facilities to be able to grow E. coli and damn it I want to use them!
Saccharomyces cerevisiae (Yeast) – I know a guy who grows yeast for his experiments and I’m sure it wouldn’t be a stretch to get him to do so in deuterium depeleted water.
So the idea would be to try to grow these in regular water and in deuterium depleted water (no ), and in the case of E. coli and yeast, perhaps in pure because I don’t think those experiments have been carried out yet. Hopefully I will be able to conclusively state whether or not life has developed a need/use for which would be a very interesting discovery indeed!
This is a little backwards because I’m announcing some preliminary results of a project that I haven’t even discussed yet. It’s also a little awkward because I still don’t know what to call this project (at least in a way that would fit in a blog post) and I have even less of a clue what to categorize this project as. With all that said, I say let’s forget all that and just get into what we all came here for:
The results!
I put some tobacco seeds in different water buffers (types of water?). My next post will explain all this, but the short is I have seeds in tap water, filtered 18MΩ (deionized water), and deuterium depleted water (water contains , but it also contains a hydrogen isotope called Deuterium that can form ). The lab has been working with a lot and found (along with research done in the 1930’s) that life is greatly hindered under pure . So Koch has been thinking that life evolved to find a use for . The hypothesis is that the seeds won’t grow as fast in water that has no compared to regular water. Let’s see what happened in the first trial of this experiment.
Day 1 of Seed Growth
Seeds in DI Water
Seeds in Tap Water
Seeds in Deuterium Depleted Water
High Res of Seeds in Deuterium Depleted Water
Now remember these are preliminary results so I don’t want to make claims that aren’t accurate, but it appears that the roots have sprouted tiny offshoots in the Deuterium Depleted water (DD water). The other option is that it is mold, I’m not sure. Hopefully this will be more clear in a few days.
Here are the results of the reaction that I posted earlier today. In lane 1 is a 1kb DNA ladder. The second visible band from the bottom is 1kb in length. The next band is 1.5kb. Lanes 2-6 are each 10ul (mixed with 2ul of 6x loading buffer) from their respective PCR reaction tubes (all the same reaction). Lane 2 clearly worked the best, while lane 3 apparently contained a failed reaction. The other lanes (4-6) worked well enough in this test run. And as you can see, since the band travel distance is right between the 1kb and 1.5kb mark, that puts these fragments at right around 1.1kb (primers annealed at ~980 and 2008 on the template strand). More tests will need to be conducted in the future, but the preliminary results appear quite promising.
. In previous experiments and 
, but it also contains a hydrogen isotope called Deuterium that can form 
