Author Archives: Josh Perfetto

Update on the OpenPCR project

It’s been way too long since the last post, so wanted to give everyone an update on the project, and where things are going.

First, despite not having a lot of updates on the blog :), I am amazed at the usage growth in OpenPCRs. I think things have gotten to the point where many people are encountering OpenPCRs in other peoples’ labs, hearing that they work, and then wanting their own.

I am also extremely pleased that it is increasingly used in education. For example, Arizona State University used 16 OpenPCRs in a Biomedical Engineering class, where students used them in conjunction with a fluorimeter to amplify and detect DNA. Even better, each group of students then designed and documented their ideas for improvements in the OpenPCR. It was great seeing the open source/kit nature of the OpenPCR being integrated into the class, rather than having a thermalcycler be simply a black box.

One thing we have learned over the course of the OpenPCR project is that despite many of their great attributes, open source projects are not necessarily the best way to manufacture and deliver physical products, and in days past kit orders were plagued with production delays. So I am pleased that Chai has taken on the physical manufacturing and distribution of kits. This has resulted in simple improvements, such as consistent shipping within 1 business day, cheaper international shipping with faster customs clearance (about 50% of OpenPCRs ship internationally), and more reliable sourcing of components. While these details are certainly unglamorous, they are truly important in getting more OpenPCRs into the wild, and thus more users of this project. Chai will also help sponsor and fund further work on the OpenPCR project.

So with the original design complete and kits being delivered, what is the future of the project? Certainly improved version(s) of the machine can be made, and this is likely to happen. But I’d also like for the OpenPCR project to be not just about PCR machine kits, but to more generally be about openness in PCR, including for example protocols, reagents, and open source analysis software. Look for more developments here in the months to come.

Alas! OpenPCR is pre-stocked

After many months of effort, we’ve finally ramped up our manufacturing to the point that OpenPCR is no longer backordered. With our latest weekly shipment, pictured above, all placed orders have finally been fulfilled, and we’re now shipping new orders within 1-2 business days. So no more long waits for your OpenPCR :)

With these delays behind us, we’re now able to concentrate on improving OpenPCR, and will have some exciting software improvements coming soon. Say tuned!

Note that while individual orders will now be fulfilled in 1-2 days, distributors placing bulk orders will continue to receive 4 week shipping quotes, until supply can be built up a bit further.

Ramping up Manufacturing: Now shipping in 1-2 weeks

It’s been no secret that the OpenPCR has been hard to get this past year. Every time we did a manufacturing run, we produced more than the last, but by the time each run was produced, the units of that run had already sold out, resulting in OpenPCR being continuously backordered. Thanks to the great community support we have received, we were able to produce 100 units in our last run, and OpenPCR is now shipping on average 1-2 weeks after being ordered. Our next run will be for 200 units, and we hope that after completed, OpenPCR will finally be pre-stocked, shipping immediately after orders are placed. I want to thank many of your for your patience over the past months as we’ve ramped this up, and hope we don’t have to count on your patience much longer :)

We’ve learned a lot over the past year about manufacturing. Mainly that it’s not much more difficult to produce 50 units instead of 25, or 100 instead of 50. We designed OpenPCR with the philosophy that we should use as many off-the-shelf components as possible, but that has come back to bite us as certain key components became unavailable from our suppliers, necessitating costly re-sourcing. But OpenPCR has grown up a lot this past year as such mistakes are corrected, and scalable manufacturing is now in sight. Stay tuned…

OpenPCR Boards Assembled

As we didn’t want to wait for professional PCB assembly, we assembled the first batch of 35 OpenPCR boards ourselves. It took some time but came out decently. The biggest difficulty was soldering the H-Bridge IC, which has three large power connections on the bottom of the package, plus fine pins all around it. We managed that by effectively doing reflow soldering with solder paste and a toaster oven, and then soldering the through-hole components afterwards.



That's one large Arduino shield

That's one large Arduino shield

As much fun as that was, I’ll let a pick and place robot do it next time. I’ve been looking at Screaming Circuits for PCB assembly in the ~100 unit range, though please feel free to suggest any other companies in the comments.

First OpenPCR kit assembled

It’s no secret that we’re fast approaching the shipment of our Kickstarter and pre-order OpenPCR kits. But today marked a new milestone: the first assembly of an OpenPCR by someone other than Tito or myself. Eri Gentry was kind enough to spend the day assembling an OpenPCR and giving feedback on our instructions, which we’ll role into the final printing. Here she shows off the completed unit, which will be housed at the BioCurious hackerspace:

The boxes you see in the background are OpenPCR components for the first kits. We’re just awaiting one more shipment from China (expected next week), and making some final software changes, and then these boxes are out the door.

Help Fund the Synthetic Bio Documentary

Sam Gaty and George Costakis have been shooting footage for a Synthetic Biology documentary over the past year or so, interviewing everyone in this emerging field, including leading synthetic biology researchers as well as DIYbio practitioners. They’ve now started a Kickstarter project to get their film edited in time for submission to Sundance 2012. Check it out, and see the great preview at the end of their Kickstarter video.

It Lives

The below is a gel showing PCR reactions from the final OpenPCR prototype that will be used for our kit production. This experiment was done to validate various polymerases rather than test OpenPCR, but thought you may want to see it. Lane 1 at the right is a control with no polymerase. Lanes 2 – 4 show amplification of a ~1kb amplicon with different polymerases/salt concentrations. Lanes 3-4 have a different loading dye, but pay attention to the fluorescent DNA band (imaged with GelGreen / Invitrogen Safe Imager).

OpenPCR PCR machine results

This is just an early glimpse – when we release the final machine kit, we’ll include a gel image with well-by-well results for an identical reaction. That release is thankfully getting closer – over the weekend Tito and I tested a prototype with full mechanical assembly and got excellent results, so we’re just getting the final pieces into production. Stay tuned for the official release date!

November Design Update

We’ve made a lot of changes recently as we near our release, so I wanted to give a quick update on where things stand.

We’ve moved to using a silicone/kapton (pending testing) heater with an integrated thermistor for the heated lid so as to increase the ease of assembly & reliability. We’ve selected a copper heat block for the wells which has excellent thermal properties (high thermal conductivity and low specific heat), and is plated in chrome to protect the copper. We’ve placed the thermistor which measures heat block temperature inside the block to more accurately measure the block temperature, which has improved the thermal control dramatically. We’ve also optimized the air flow through the case/power supply to cool the heat sink more rapidly, thus improving cooling performance.

We’ve made a few changes to aid ease of assembly of our kits. We’ll be including an ATX power supply which has all cables other than the 24 pin motherboard connector removed to save room in the case. Due to the amount of surface mount components on our board which can be tricky to solder, we’ll be including pre-assembled circuit boards as part of our kit. We’ve also moved to the newer Arduino Uno, and are currently researching how we might leverage the new USB capabilities that it provides.

Tito and I are hard at work sourcing all the materials for our kit, which we plan on shipping mid-December. We have large lead times on some of the more custom or hard-to-find components, so our priority right now is getting our orders in. As such, some tasks such as documenting the design & assembly instructions have taken a back seat for the moment, but rest assured that when we begin shipping kits, there will be complete open source designs/bill of materials on our website for those of you that want to hack on your own. We’ve also started accepting pre-orders for our kits if you want to be assured of getting one of the first batch of units. Kits will include all the parts you need to build your own OpenPCR – you’ll just need some basic tools like screwdrivers.

We have plans for some great new things once we finally get past our OpenPCR ship date — stay tuned!

The Importance of Speed in PCR

The job of a thermocycler is to implement a set of temperature changes in a reaction volume. The biological requirement for PCR is to implement a set of temperature changes such as this:

Repeat 30 times:

  1. Denature at 95 C for 30 seconds
  2. Anneal at 64 C for 45 seconds
  3. Extend at 73 C for 45 seconds

If you add up the times required, the biological requirements of this reaction take 50 minutes. However it also takes time for the PCR machine to transition between each set of temperatures, which is effectively wasted time. Here’s where the speed of the PCR machine comes in. If the PCR machine is capable of changing 2 C/s, then starting from 20 C the above reaction would take 66 minutes. However if it was only capable of changing 0.5 C/s, the above would take nearly twice as long: 114 minutes.

When we sat down to design a low cost thermocycler, we had options that were cheap but slow, such as relying on convection and radiation to cool the heat block. We opted instead to go with a more complex peltier cooling solution so the machine would be faster: our prototype currently ramps at about 2 C/s.

Why did we feel speed was so important? There’s two reasons.

My formal background is in computer science. When writing new software code, I do not write it correctly the first time. I may have a design flaw, or simply have made some mistakes. However I’m able to get rapid feedback, correct the issue, and test it again, so making mistakes is not a problem at all. In software, it’s typically possible to get feedback in a time ranging from seconds for small changes to at most about 10 minutes to run a well designed test suite on a modestly sized application. That rapid feedback cycle not only allows engineering projects to proceed rapidly, but it is also a large part of how the novice developer learns the art of software development.

When I work in biology, my experience is anything but that. Experiments (or design-build-test cycles) run anywhere from hours for simple operations, to days if cells must be grown, to weeks if DNA must be synthesized, to years for long-running clinical trials. Faster PCR cannot solve all of that, but it can do its part for the experiments where it is critical.

For example, I’ve been working to create a SNP genotyping protocol which would allow people to read any SNP of interest from their own DNA. It’s complex and involves DNA extraction, PCR, and a gel run. Currently it takes the better part of a day to complete; reducing it so it could be run twice a day would be a big win, especially considering someone new to biology is likely to make mistakes the first couple times. We should all be thinking about how we can make design-build-test cycles faster when we create tools for biology.

The second reason is a little more technical. The PCR reaction makes use of a “thermostable” polymerase enzyme, which really means that the polymerase doesn’t denature and cease to function right away at the denaturing temperatures of PCR. In actuality the polymerase has a half-life which is reduced as temperature increases. At the end of a 25 cycle PCR run, the majority of the polymerase may in fact be destroyed, which is a limiting factor in the amount of amplification possible.

That can be a problem if you’re starting from a very small amount of DNA, as may be the case in a forensics application. It is also important if your upstream DNA preparation protocol is sub-par. For example, suppose a DIYbio enthusiast was doing sushi DNA barcoding. While they have access to an enormous amount of DNA to begin with, they may use a lower-cost DNA extraction method which doesn’t remove as many PCR inhibitors as a Qiagen extraction kit would. That’s not a problem so long as you’re still able to get enough amplification for sequencing, which you can do by extending the number of PCR cycles so long as you have enough working polymerase left.

So faster PCR can introduce resilience to overcome poorer upstream processing, and if that enables you to get the same results with cheaper reagents, that’s another win for everyone.