Who is familiar with using thermal cyclers in a bio lab?

Your lab should have an up to date SOP or at least a manual for the proper operation and maintenance of your “CCC”. If not then I would consult the manufacturer for advice.

Yeah, you really shouldn’t be doing anything you haven’t done before without a procedure. It’s entirely possible the thermocycle has an end ‘wait’ segment where it simply cools the tubes until you come to get them, so you don’t have to watch it and grab it just as it ends.

In general, a standard PCR will take ~3–5 hours, depending on cycling conditions.

@gondwanalon And of course we don’t. The thing is, the CCC program was made up by someone. It’s not an official program that comes with the cycler. Someone, a long time ago, created it for this specific procedure. The other annoying thing is that when I look back to see how my predecessors did it NOBODY BOTHERED to mention exactly what the CCC program entails and how long it takes. Which is seriously fucking irritating.

Is there no way to examine the program specifics on the machine?

Are you working in a medical research lab? Perhaps you can locate the notes that your predecessor generated to find the information that you need. Or, if you have the time then you may be able to experiment and develop your own procedure that works.

Yes, I already examined the program’s specifics on the machine. I just said that the machine said it lasted around 22 hours. That makes no sense being that my predecessors have done it in a day (as in 9AM-5PM). It’s not medical research but research nonetheless, and every time I find someone who did the procedure, they just label the step as “Set thermal cycler to CCC” without actually stating what CCC entailed or for how long it lasted.

Well, does it give you any other info? Usually the machine should somehow break it down into the distinct stages, if we have that we can figure out why it’s taking 22 hours.

Like a typical program on my machine would display as

1: 95 deg 5 min
2: 95 deg 30 sec
3: 55 deg 30 sec
4: 72 deg 1:30
5: goto 2 29 times
6: 72 deg 5 min
7: 4 deg forever

It has three steps.

First step is supposedly 22 hours at 37 deg C. I was thinking what if someone accidentally set it to 22 hours when they meant 22 minutes?

Second step is 20 minutes at 68 deg C

Third step is 15 minutes at 4 deg C

And then it ends.

That’s… not PCR. What’s in the cocktail that you’re adding, other then the DNA?

PstI enzyme, BSA 100X and NEBuffer #3 10X (New England BioLabs), and reagent grade water

Ah, okay. So this is your digestion. The 22 hours is certainly excessive (and actually can lead to bad results), but 22 minutes is rather short. A digestion should normally proceed for ~1–3 hours. The first 37 deg segment is where the enzyme is active, the second at 68 deg deactivates the enzyme, and the last just cools it down (not sure why it’s just 15 minutes, but it’s not all that important). I’d suggest changing the first section to ~1–3 hours (depending on how sure you need to be that it’s 100% cut) and then you should be good.