Thursday, 10 August 2017

Diagnosing thermocycler issues with a cheap thermocouple and logger

Occasionally I have a peek at /r/labrats, which is a subreddit for people who work in science labs. One of the regular themes is the bemoaning of the unpredictable nature of research shared by all scientists everywhere, and such was the thread that inspired this post.

One user was complaining about some dodgy amplification results, cursing the capricious nature of the PCR gods, and among the suggestions for possible contributing factors was possible cold spots in the thermocycler. The original poster was a bit doubtful about this, so I thought I would link them to a blog post I wrote on this very topic a year or two earlier. However this was slightly hampered by that fact that it turns out I hadn’t actually ever written the post – I just meant to, and then presumably forgot to. So, in reference to that thread (note that while that initial reply has since been deleted, the subsequent thread with my replies remains here), and with future similar occasions in mind, here’s some of what I intended to say. It’s two years late, and I’m in a different institution now without access to all the files I had then, but hopefully it could still be useful.

Essentially, I was in a somewhat similar position to that Reddit poster, in that I had been getting some dodgy and inexplicable PCR results, as had others in my lab. After a series of control experiments, I began to suspect that it was the potentially the cycler itself that was to blame – the four-block G-STORM I was using was getting pretty old (the cream plastic was even starting to go that grungy ‘old keyboard’ shade of yellow), so I resolved to try to measure whether it was operating as expected.

To do this I ordered a cheap K type thermocouple probe and a basic logger (which, combining my memory with my Google Fu skills, I think was the EasyLog EL-USB-TC-LCD Data Logger from Lascar Electronics.

What I did then was poke a small hole in the top of a 0.2 ml PCR tube, the same kind I use for all my PCRs, add 50 ul of water inside, poke the thermocouple in through the side so that the probe tip is under the water level, and then hold it in place/seal the hole with a tiny strip of Parafilm. I don’t have a photo, but I found this beautiful reconstruction that I drew for a lab presentation I gave around the time:


Then I simply ran a bunch of different cycling steps, some PCRs, some incubations, some mock runs, and then plotted what temperatures were recorded throughout these programs, for different wells in the same block, across the four blocks in the machine. Unfortunately, I don’t seem to have copies of most of these plots, but the few I do have make the case well enough. Here are the first tests, showing that different opposite corners of the blocks failed to hit the proclaimed temperature in simple two-step heating experiments:

And here is a far more damning example, of two wells in the same block of the same PCR machine (which had incidentally recently been ‘repaired’) in a short mock PCR cycle (denaturing at 95°, annealing at 55° and then extending at 72°): one of the wells sampled failed to hit either denaturation or annealing in the allotted time! No wonder these amplifications were failing.


Happily, in this situation, it was a problem easily solved: we bought a new PCR machine, and I’d caught the problem before any important results could be affected. However, it really does make you wonder just how much you can trust your thermocycler? Or your heat blocks (especially above 60°, where the alcohol thermometers found in labs cease to be useful)? You know, those crucial bits of kit upon which whole sections of the output of your lab is possibly utterly reliant, and yet which you probably have no readout for or data on other than the traces that the inbuilt software displays on the screen (which I should say, were always completely normal on all of the dodgy blocks I tested).

The total price of the kit required to do the tests I described above was about £80, or $100, with 95% of that being the logger. This is considerably less than you probably regularly spend on polymerase, if your lab does a lot of PCR, which seems like a pretty small price to pay to be confident your reactions are proceeding as planned.