Thermal Cycler
We have a Perkin Elmer Thermal Cycler 480. It's a bit of a monster, but it works :).
It has a heating block set up for up to 64 microcentrifuge tubes, each sized for a 0.5ml microcentrifuge tube. It doesn't have a heated lid (but building one shouldn't be too hard) but until then we can just go old skool and use mineral oil.
Just above the screws, there's a big sticker saying "Do Not Open". Hence, [here's a picture of the machine's internals]. At the top of the image, the black square is the underside of the heating/cooling block. Most of the volume is a compressor for the cooling, with the insulated cooling piped clearly visible (Most modern machines are based on Peltier systems, and hence a fraction of the size of this one). At the back of the machine there's a serial port, but this seems to just be a printer port rather than allowing any external control.
The UI is, er, interesting. After spending some time with the manual I think I have it figured out, but it's definitely something that's going to need training and / or practice to learn to use. Over time, we should try to build up a cheat sheet on this page.
Upgrades: If someone can build a heated lid, it could save us a degree of faff later on. Basically, it would be a metal block heated to approx 95 degrees (+/- 5 degrees should be fine) that rests on the lids of the tubes while the machine is running.
How to use it:
I strongly suggest you RTFM before following these instructions. Even if they're 100% correct, it's easy to get lost in the UI. The interface is quite logical and comprehensible when you're used to it, but it's bafflingly opaque to first-time users! The manual is stored in the lab - read it if you want to find out how to chain programs together to create a method.
Setting parameters from scratch:
Soak (keep at a constant temperature):
Start with the UI saying "STEP to enter file 1" (or file 4, or whatever).
- Press File - 1 - Enter
- Enter temperature and press Enter
- Press YES to start the program.
- To pause the soak, press NO. Press it again to stop the soak.
PCR:
- Press File - 4 - Enter
- Enter temperature for denaturation step and press Enter. Press Clear if you make a mistake.
- Enter time for denaturation step in minutes (enter) and seconds (enter).
- Enter temperature for annealing step
- Enter time for annealing step
- Enter temperature for extension step
- Enter time for extension step
- For 4th step enter 0°C and 0 time to signal end of process
- Select "no" for "Auto segment extension" (the cursor should be on No by default, so just press Enter).
- Enter number of cycles and press Enter.
- For "link to stored file", you can do one of two things. If you enter 0, the thermocycler will shut off heating and cooling to the block after PCR, which means it will gradually fall to room temperature. Alternatively, if you enter 20, the thermocycler will run a 4 degree soak file after PCR -- equivalent to putting the samples in a refrigerator. (The magic number 20 refers to a soak program we previously saved. Don't overwrite it!)
Running stored files:
- To run a stored file, press File -> [file number] -> Enter -> Start
- If you want to run a method, just run the first file and it will run the whole method for you
Stored files:
- Initial D - 95C 5 mins, then 35 cycles of D-96C 1 min, A-55C 30 secs, E-72C 1 min.
- File 60: 96C 5 mins (Initial denaturation) - linked to file 61
- File 61: 35 cycles of D - 96C 1min A - 55C 30s E - 72C 1min - linked to file 63
- File 63: Soak at 4C
Incubation program for restriction digest:
TBA
