Today I spent 7 or so hours in the lab, trying my darndest to have a pretty looking PCR product, I tried to be very detailed double checking if i pipetted everyting correctly, trying to keep components well mixed and...
It was all for nothing, the gel looks like shit, I am 90% sure the cyclecount was too low (twice, before the second time I mentioned it to my supervisor but she told me it will be fine if I pipette things right, I get were she was coming from, the PCR was optimised and was clean before), the band of small nucleotide (primer dimers) products is really overpowering. Maybe annealing temp is too low too, tough stuff, the other worrying thing is that actin2 band (housekeeping gene to compare to) was inconsistant (It should be the same!).
It's fiddly and slow, there should be some other way to do molecular biology, I do think cartesian 3d printer-like pipetting machines are really cool (not like I have access to those right not, just pondering) but after doing it myself for a while and how mixing is wierd and pipettes also can be wierd I see the engineering challenges in that.
I even made a recording of myself pipetting all the things to be 100% sure I added everyting in quantities it should be added in, I believe cDNA is done properly too, so I'd assume it's something with the PCR. but it's not like the PCR can change one actin product without changing another.
None, tommorow I don't have anything planned I will catch up on the yesterday's paper and read a new one.
I was too tired, hadn't slept enough, hadn't used enough caffeine, didn't use music to ward off fatigue, went home back after the lab, ate some frozen curry I had and instead of caffeinating and napping for 1h, just slept for 4, then got up watched some youtube (new Kruggsmash!!) and went to sleep again, the tea in my advent calendar was a boring old chamomile (first three days were great, earl grey, sebcha green and imitation yerba).