Testing my PCR thermocycler, Part I: Setup
Way too long ago, I bought a used thermocycler on ebay. It's quite a monster, a Perkin Elmer Cetus, and I've been meaning to test it. I initially thought about testing it simply by running a PCR and seeing whether I get the expected results, but there are so many sources of error in this plan that I've discarded it. There are just so many things that can go wrong with a PCR, even if the thermocycler works perfectly - not least due to my very limited lab skills.
So instead, I'm going to measure the temperature of the heating block over a few cycles. There are even sources on the internet that document the temperature profiles of other thermocyclers, so I can use those as a reference. All in all, this approach does not only yield quantifiable results, but seems more attainable to me. Plus, this will (ideally) produce a setup that allows me to test other thermocyclers, too.
So. After some investigation of temperature sensors in general and in particular, I decided to use an IC. There are some that are accurate enough for this purpose in the required temperature range, and I wanted to spare myself the hassle of constructing a circuit for an NTC, calibrating it etc. I selected the Dallas Semiconductors DS1621S, which comes in an SOIC8 package. Have a look at the data sheet, if you're interested. The device gets stuffed into 0.5 ml PCR tube, with the wires protruding out the top, like this:
The Perkin Elmer Cetus doesn't have a heated lid, so just leaving the lid open for the wires shouldn't distort the temperature readings too much.
The DS1621S gets hooked up to an Arduino and the data collected on my computer.
After soldering, I realized that I won't be needing all eight pins, but alas. If the data from the initial run make any sense at all, I'll prepare more of these sensor tubes, so as to measure temperature across different positions on the heating block. But now, on to the actual measurements. Data will be made into pretty graphs and posted here once I'm done.
So instead, I'm going to measure the temperature of the heating block over a few cycles. There are even sources on the internet that document the temperature profiles of other thermocyclers, so I can use those as a reference. All in all, this approach does not only yield quantifiable results, but seems more attainable to me. Plus, this will (ideally) produce a setup that allows me to test other thermocyclers, too.
So. After some investigation of temperature sensors in general and in particular, I decided to use an IC. There are some that are accurate enough for this purpose in the required temperature range, and I wanted to spare myself the hassle of constructing a circuit for an NTC, calibrating it etc. I selected the Dallas Semiconductors DS1621S, which comes in an SOIC8 package. Have a look at the data sheet, if you're interested. The device gets stuffed into 0.5 ml PCR tube, with the wires protruding out the top, like this:
The Perkin Elmer Cetus doesn't have a heated lid, so just leaving the lid open for the wires shouldn't distort the temperature readings too much.
The DS1621S gets hooked up to an Arduino and the data collected on my computer.
After soldering, I realized that I won't be needing all eight pins, but alas. If the data from the initial run make any sense at all, I'll prepare more of these sensor tubes, so as to measure temperature across different positions on the heating block. But now, on to the actual measurements. Data will be made into pretty graphs and posted here once I'm done.
Lisa --
ReplyDeleteLooks like a cool idea, interested to see how it works out!
I'm building an open source thermal cycler and having an easy method to test that it works would be great. Keep in touch :)
Tito
Hi
ReplyDeleteI don't know how familiar you are with thermocyclers so you may have already thought of this - the IC/ thermometer probe needs to be immersed in liquid for correct temperature sensing (to replicate PCR conditions and to provide thermal conduction). Your IC won't like PCR buffer but should survive being immersed in oil. The IC is also pretty large so it may stick out of the liquid a bit as well as conducting heat through the wires. It's certainly not much good high up in the tube as it is in the picture. Does the lid close? If not then there's another problem. You'd be better to get one of those tiny little thermocouples, calibrate it against another thermometer if necessary (eg your DS1621S) and use that to sense the temperature in a liquid volume the same as you'd be using for PCR (eg we use 10, 20, 50ul). The Cetus uses 0.5ml tubes right? So it may need larger liquid volumes - 100-200ul maybe. With the IC where it is now you'd have to just about fill the whole tube - not ideal.
I suggest you get a cheap digital thermometer or multimeter with thermometer function and a K type thermocouple and use that. They are not very expensive. You may not be able to log data with it straight away but at least you're getting accurate readings.
Good luck!
Benjamin
@Benjamin/Anonymous:
ReplyDeleteI'll keep all that in mind for later.
As far as the Cetus goes, I ended up using a digital thermometer as a reference, with the probe immersed in water in the (0.5ml) tube - because I didn't trust my setup with the DS1621 very much when I got the first readings. However, the digital thermometer confirmed that readings were ballpark-accurate at least, which was enough to indicate that the old Cetus didn't even come close to the target temperature. It wouldn't go over around 60 degrees Celsius.
Hm. But reading your input again, I'm not so sure I didn't simply royally mess up the temperature measurements. I'll give it another go when I find the time. And yes, the Cetus essentially has an open lid - it's just a piece of clear plastic to keep the user from accidentally touching the heating block during operation.
Anyway, I've actually bought a new thermocycler in the meantime, since I figured that I'll be making enough mistakes on my own, and I really don't need additional errors introduced by crucial but dodgy equipment.