When I was in my teens, the thing that I loved more about science was the opportunity to learn something new every day. I loved that what I thought I knew was never exactly true, and hated it at the same time. I wanted to know what we’d be told in our GCSE classes, or our A-level classes, or our undergraduate lectures long before I was old enough for it to be on the syllabus. I had this idea that if I kept studying science for long enough, eventually it would make sense.
It will never make sense.
Today I have sat down for some number crunching and had to admit that there is something fundamentally wrong with my qPCR. I have three single deletion lines, and one double deletion line. Each single deletion line corresponds to one copy of a particular gene. I have primers for two of those genomes. The expectation therefore would be, that when you test those primers against genomic DNA from the deletion lines:
Deletion Line 1, missing Genome 1, would amplify with only primer set 2
Deletion Line 2, missing Genome 2, would amplify with only primer set 1
Deletion Line 3, missing Genome 3, would look like the wild type line
Deletion Line 4, missing Genomes 1 and 2 would not amplify at all
Except… they don’t look like that at all. I know that Genomes 2 and 3 are very similar, which explains why the double deletion line amplifies well with primer set 2, though not with 1 at all. And similarly, Deletion Line 2 is likely to have some amplification with primer set 2, because it still has Genome 3. But there is no logical reason why Deletion Line 1 should have any amplification with primer set 1: never mind anything close to equal amplification!
I am confused. And frustrated. And annoyed because I really wanted to get this data on my poster for PAG. I would quite like to take the day off and sulk, but there is way too much that needs doing, and I had a day off yesterday to look after a sick friend so I absolutely cannot afford the time.
And in a moment of spectacular stupidity, I paid off the wrong credit card (the one that wouldn’t have automatically cleared until after my next stipend payment arrived) meaning that I will now be surviving until January 1st on about £70. And I haven’t bought the family Christmas presents yet.
Today is not my day.