This Instructable will describe how to make a PCR Thermocycler for Field or Teaching applications, from scratch, using a commercial temperature controller, a temperature probe, a PC cooling fan and basic shop tools.
The unit performs reliably, is menu driven for programming various PCR (Polymerase Chain Reaction) "recipes" (so you don't have to use a computer but there is free downloadable software that will let you do this and we provide the PIN diagram for making the cable). It cycles temperatures at 0.15 degree centigrade per minute. The difference in actual temperature (Process variable) from "control" temperature (Set variable) varies about 5 degree centigrade. That means we set the temperature at 5 degrees higher than the temperature we want to achieve. This is due to the bias in the thermocouple probe.
In a Nutshell: We use a piece of aluminum rod, drill a 1/4" hole in the center of one end for a cartridge heater and another hole near the rim of the same end for a thermocouple (temperature sensor). Additional holes in the same end of the rod are for sample tubes (0.2 ml eppendorf tubes).
This "heating block" is positioned on a small bracket over a PC cooling fan. Both are placed inside of a metal coffee cup. A commercial temperature controller ( available from Omega Engineering) is used to control the heating and cooling cycles. End of story.
It reaches and holds (soaks) at various temperatures and can also be used for "In situ" Hybridization protocols. It can be programmed from the face plate of the controller or connected to a computer by a serial cable. The control software is a free download.
Project Team:
R. Siderits
A. Marcus
F. Ivalde
A. Andoh
M. Swift
O. Ouattara
W. Lecorchick
S. Singh
The unit performs reliably, is menu driven for programming various PCR (Polymerase Chain Reaction) "recipes" (so you don't have to use a computer but there is free downloadable software that will let you do this and we provide the PIN diagram for making the cable). It cycles temperatures at 0.15 degree centigrade per minute. The difference in actual temperature (Process variable) from "control" temperature (Set variable) varies about 5 degree centigrade. That means we set the temperature at 5 degrees higher than the temperature we want to achieve. This is due to the bias in the thermocouple probe.
In a Nutshell: We use a piece of aluminum rod, drill a 1/4" hole in the center of one end for a cartridge heater and another hole near the rim of the same end for a thermocouple (temperature sensor). Additional holes in the same end of the rod are for sample tubes (0.2 ml eppendorf tubes).
This "heating block" is positioned on a small bracket over a PC cooling fan. Both are placed inside of a metal coffee cup. A commercial temperature controller ( available from Omega Engineering) is used to control the heating and cooling cycles. End of story.
It reaches and holds (soaks) at various temperatures and can also be used for "In situ" Hybridization protocols. It can be programmed from the face plate of the controller or connected to a computer by a serial cable. The control software is a free download.
Project Team:
R. Siderits
A. Marcus
F. Ivalde
A. Andoh
M. Swift
O. Ouattara
W. Lecorchick
S. Singh
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Signing UpStep 1: What is a Thermocycler?
What is a Thermocycler?
A thermocycler raises and lowers temperature. For molecular or DNA applications it needs to heat up to about 90-95 degrees centigrade. DNA which usually exists in double strands in a helix (like a spiral staircase) and at about 92 degrees centigrade the strands will break and fall apart.
The solution that the DNA sample is in has extra "bases" that will connect to the broken rungs of the ladder to make two new ladders. To do this part we need to cool down the sample to the point where "bases" can connect to the separated DNA strands. If you cycle through this temperature range from 20-30 times you can make millions of copies of the original DNA.
So in summary, a thermocycler can make a million copies of one original piece of DNA. These copies can then be used for other types of research. The prototype that we build can take 6 sample tubes (0.2 ml eppendorf tubes). There is room for one blank, the temperature probe and a central cartridge heater.
Learning sites that are excellent:
- DNA Extraction Online Tutorial
- Home DNA extarction of DNA (Instructables)
- PCR
- PCR Song (must hear)
- PCR Protocols
A thermocycler raises and lowers temperature. For molecular or DNA applications it needs to heat up to about 90-95 degrees centigrade. DNA which usually exists in double strands in a helix (like a spiral staircase) and at about 92 degrees centigrade the strands will break and fall apart.
The solution that the DNA sample is in has extra "bases" that will connect to the broken rungs of the ladder to make two new ladders. To do this part we need to cool down the sample to the point where "bases" can connect to the separated DNA strands. If you cycle through this temperature range from 20-30 times you can make millions of copies of the original DNA.
So in summary, a thermocycler can make a million copies of one original piece of DNA. These copies can then be used for other types of research. The prototype that we build can take 6 sample tubes (0.2 ml eppendorf tubes). There is room for one blank, the temperature probe and a central cartridge heater.
Learning sites that are excellent:
- DNA Extraction Online Tutorial
- Home DNA extarction of DNA (Instructables)
- PCR
- PCR Song (must hear)
- PCR Protocols
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First you need to heat up a DNA sample to 100 degrees centigrade. You might think that would be impossible but I used two minco thermofoil adhesive backed 1 inch circular heaters wired to two USB ports. Now you need to have a switch turn them on and off so I used a phidgets.com dual solid state relay connected to a phidgets: 8 analog in/8digital in/8digital out board. Now you need to sense the temperature so that you can turn the heaters on and off at the right time. I used a phidgets temperature sensor with a J type thermocouple. OK so now you have the temperature side down, how about the light side. I used a photoresister to monitor the light level in the sample chamber and brought that into the phidget board on another analog channel.
No you only need two more things. A light source the emits at a blue light (I used an ultra bright LED at 480-490nm on the second of the dual Solid State Relays) to excite the SyberGreen when it binds to double stranded DNA and a band pass filter to only let a narrow band of green light through to the photoresistor. The band pass filter was from Fisher scientific. I wrote the interface to sense temp and light and to control the relays for heating with LABVIEW.
Long story short, I've got an essentially disposable RT-PCR platform that works on USB power only and costs less than 300$ total cost. You can stick it onto the back of any standard Notebook computer. This enable field research at a fraction of the cost of a small RT-PCR platform. For what its worth, you can run it from a remote as well.
I was told (often) that you can't heat something to 100 degrees centigrade (denaturation temp for DNA) with a USB (4.5 Volts and 100 mA). Turns out its really easy. We used two adhesive backed 1" diameter Minco Thermofoils on separate USB ports. A quick program in Labview to monitor temperature from a thermal probe, a styrofoam thermal chamber to prevent heat loss and a USB Phidget relay switch to control the heaters made it work to commercial nearly specs. We can control, soak, cycle and ramp with no problem.
We then started on the Real Time component using a 1$ "teal" LED and a UV LED to excite Sybr green and a cheap 1 cm (25$) band pass filter from Edmond Scientific to select out the emission wavelength for "Sybr greener". A 50 cent photoresistor should give us the ability to detect amplification in real time for under 200$.
That's where we're at now.
Thanks for the kind words; We just printed our our chamber on an UP3D! printer (see attached image) . It cost 11 cents. The front and back panels hold the adhesive backed Minco Thermofoils (as the USB powered heat source). One set of wires in the image is the pressure fit Aqua LED with the perfect wavelength to excite Sybr green, we also now have a second LED in UV range. The other side holds the Band Pass filter in front of the photoresister or photodiode. Its really easy to swap out LEDs and bandpass filters to fit your needs. All controlled with USB "Phidgets" (SSR, 222 controller and Temperature sensor) and interfaced quickly with Labview graphical programming platform. Good weekend project. Its essentially disposable, or you could have several in a row, etc... As a matter of fact you could actually run it off of a solar panel and control it with a 9 volt battery powered microcontroller, even remote the data over wireless (or just log it).
We're now tweaking aspect of the insultation, when time permits.
We've considered using a sinmple LED display on a cheap Single Board Computer (SBC) also from Phidgets. Most of the "solar" work was done by the 10$ medical centrifuge team (see one of our other Instructables for that one. Great idea, used empty bullet casings as buckets and celophane for virtual tubes. Anyway, here are the filters we used.
Edmund Scientific Filter INT 535nm 11.80 mm diameter item number 43071, ordered a second type with a slightly different wavelength pass-through (532 nm item number 43070). We wanted to make sure that we dealt with any secondary peak issues.
any advice on how to go about this?
Just use a hack saw and a hand drill.
You can use a square piece of stock with holes drilled at a distance equally placed from the center heating cartridge. Then cut "fins" to enhance cooling and approximate a round shape.
You can also use the hack saw to take the corners off the square block. You might even consider scribing an octagon on the surface and cutting a square block down to an octagon shape to drill the holes into.
I used a round shape so that temperature would diffuse radially and at a constant rate while the outer surfaces are cooled by the PC fan at the bottom of the mug.
Here's another idea, cast a heating block using JB weld compound or even use the JB weld to create a custom shape from some scrap material.
Another solution would be to pick up some aluminum tubing (to hold the sample tubes) then set short segments of the aluminum tubes in a JB weld "form" to create a custom heating block.
When you get ready for the programming step using the PC let me know I'll send you a few examples. The performance is surprisingly good on this thing.
If you get stuck let me know and I'll help. By the way this is perfect for using the Quick Extract mRNA or DNA extraction kits
http://www.epibio.com/item.asp?ID=502
and yes you can make this into a real time PCR platform without much trouble.
Best regards;
R
I solved my heating head issues because the auto-tech teacher in my school had a milling machine and agreed to cut it for me.
However, when i purchased the fan my budget made me jump at the cheapest fan available which came back to be so tiny (it can't be more then 1.5 inches wide and long)
I'm currently wondering if a longer cooling period could suffice in its usage or whether i should be reading the companies return/exchange policy right now also i have the cartridge heater which fits nicely into the heating head, but has 2 wires at the end causing me to wonder how exact am i to wire this. i do not have the controller yet but will inform you immediately when it comes.
If so send address to rsiderit@rwjuhh.edu
I'd be delighted to help you get going with the program and would be glad to send you my "recipes" (that's what the program calls it's stored macros).
You will also be able to program the controller directly from the faceplate but logging the data to a text file and then importing it to an excel worksheet is fun.
It should be easy to have a pc-controlled version as mentioned above.
Ie the PC takes direct temp measurements and switches on the heater and fan to control the thermocycler directly. Hopefully this should reduce the cost dramatically while not affecting the accuracy of the device.. can you think of a way to do this, or is it imposible?.
http://gizmodo.com/5123581/make-a-usb-soldering-iron-to-build-more-crazy-usb-gadgets
Thanks for the comment!
After reading the article i looked closely at the cn8200 controller and the thermocouple, the combined cost of these devices comes to 700-800 dollars.
for a total sample size of 5 samples it not worth it plus not ever one has a machine shop at home either.
Good work, but not really practical or economically worth wile.
No enemies here.
Here's a breakdown of the low cost and higher sample number version.
We just wanted to demonstrate some control features and get some data about performance in a radial heat diffusion design.
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The basic cn8200 cost me 285$ (new from Omega electronics), you don't need the one that connects to the serial port on the computer. The basic unit is programmable from the keypad. I used the serial connect just to prove that the performance was as good as a commercial unit and to make fast changes to the program. You can get both cheaper on Ebay and remember to try www.fatfingers.com
You can also paste a silicone rubber heater to the bottom of an aluminum block that has many more sample wells (say 20). The radial heat diffusion our design will be revisited for the USB powered version. Turns out a USB powered Soldering iron with or without a 9V battery assist works just as well as the cartridge heater. Can you imagine a PCR system that runs on 2 USB ports! Anyway, the silicon rubber coated heater is 3x4 inches, is very thin and it will draw about 2-3 amps. It will heat a sample block up to 100 degrees centigrade in about 90 seconds at 6V. Just use silicon cement not thermal paste to stick it onto the block so that the silicon coated heater and the aluminum sample block stay together. The principle is the same. The thermocouple is about 20$. If you want to stay under 200$ then I'd suggest using the Phidget USB controller. The on-board rela can switch a higher rated relay but consider adding a diode to the input terminals to "reduce inductive sparking"
By the way we're about to post a 4 slide CISH IHC heated water bath that will hold slides at either 50 C or 98 C for 10 hours using a 20$ temperature controller (from MTM temperature controller
http://www.mtmscientific.com/tempkit.html ). We are also trying a high temp limit controller but we needed to CNC our own PCB - http://www.escol.com.my/Projects/Project-03%28Thermostat-1%29/Proj-03.html It will use the silicone coated heater as an example.
By the way, we're still working on the RTPCR part of this project using ultrabright LED, to excite the sybr green, a photoresistor and two band pass filters. We're using the Scratch program and a PicoBoard to monitor temperature with a thermistor bead and to take timed light readings. This would mean that anyone with a picoboard could run the RTPCR program from the internet. You would just need to get the temperature cycling with any kind of controller i.e. phidget or a completely USB powered version. One USB to power the fan, one for the heater and one for the PicoBoard. If i need robotics I'll use the Lego Wedo robotics servos the are controlled by the Scratch Program. You can even control switches with the servos, which would mean that the entire project could be run by a Scratch program that might take less than an hour to write.
Best regards
Sid
....user input ---->interpreter---->instructions written to CRON ------>comms carried out by small C programmes
just an idea for ya to ponder....
...Nice build btw! lol