When you set up PCRs, often you first make a master mix and then aliquot it into smaller portions to which you add template DNA (the stuff you need copied). Often you do one extra reaction more than you need, since you tend to run out due to pipeting error. But you don't need that much extra normally...
For the last few months, I kept on running low when aliquoting - often not having enough for the last tube. I would make 10 reactions when I really needed 8, so about 36μL extra, for 18μL aliquots. And STILL I would not have enough for all reactions. I calculated my recipe numerous times in numerous ways, and it should have worked. I tried the most careful pipeting technique I could manage, although it would be a rather exquisite accomplishment for pipeting error to be THAT bad!
I suspected the pipets, but my labmates were all like "Oh don't blame the pipets now!"
So I got sick of this. I grabbed water, and went to the analytical balance. I check the p20 - everything more or less fine. Then I grab my p200...
Yes, it only pipets ~75% of the set volume. FOR THE PAST FIVE MONTHS.
So yes, when all fails... check your pipets. Their calibration could be way off by now... (after changing all the water and buffers and stock solutions, that is.)
Now, my burning question:
How the hell did my PCRs work for the past five months despite all my concentrations being absolutely RANDOM!?
So much for molecular biology being an exact science, hahaaa!
Actually, molecular biology is voodoo. Seriously. You can make things exactly right and they don't work. You can leave your DNA out on the bench for a couple nights and completely screw everything up while pipeting... and it magically produces the bands you want. I think it depends more on the star alignments or something...
Narrow-minded, short-sighted university administrators
5 hours ago in The Phytophactor