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:
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.
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…