So qPCR is still kicking my ass. Integrated DNA Technology actually tweeted me back in response to my moaning about LNA oligos, which made me smile even if it wasn’t especially helpful.
Essentially: my primers may be awesome (provided I can acquire an unrealistic amount of DNA) or they may be unusable. I can’t tell without testing them with a stupid amount of cDNA. (Which I don’t understand, because they worked in regular PCR just fine). Continue reading
Today I became a proper postgraduate student (after 3 years two months and about five days) by purchasing Ramen noodles for the first time ala PhD comics:
Unfortunately lab work is less successful. There are about six weeks left until PAG and I desperately need to crack on and get this data. Having finally – I hoped – solved my qPCR non-specificity woes by spending inordinate amounts of money on LNA oligos, which – at 65C – became specific between homoeologues, I am rapidly finding that they are so specific in a qPCR reaction, even at 60C, that they will not make it to threshold fluorescence without using more cDNA than I can possibly afford to put in a reaction.
This is what I believe is called a Catch 22.
I am now drowning my sorrows in salty, MSG-y goodness and dreaming of a life of running routine experiments instead.