I did all the steps to get a cDNA product, starting from "noDNA" RNA I got last friday, PCR to check if there is no DNA, cDNA synthesis, PCR to check if it succeeded and I think I have the product I expect, I tried my very best to be very meticulous in pipetting things properly, despite that some bugs creeped in, I forgot (and vigilantly corrected it before it became a problem) to add crucial components of the mix (once cDNA, and once taq polymerase, it's so easy to start to daydreaming when pipetting), also I somehow I have no idea how in one mix the volume ended twice as big as I expexted it being and it completely dissapeared once I added it to a well in a gel (???).
I should have a lab-bodycam on meI am sure I botched my negative control for the final PCR, instead of pipetting water I pipetted my negative (no primers) control from cDNA synthesis (I need to think it trough how does that relate to a failed negative control will do that after I read the paper for today.) to visually confirm the cDNA synthesis by using primers targeting a reference gene (actine2), and the gene I am testing for in mutant vs. wild type.
I also somewhat botched the pipetting things onto a gel in the final gel running the pcr results, I was a little tired, but all my failings were limited enought for my work not to be in vain, I am quite confident I will get a nice looking gel tmrw.
Paper is about Arabidopsis thaliana (At) MLO family of genes, and it's very fresh, it also has cited one of the a key papers on mlo11 from (2014), and abstracts mentions root thigomomorphogenesis (touch response, a thing mlo11 is known for) and promises a broad review with some in sillico analisis on the mlo family, so I hope I will learn something from it.
Hmm hmm... mlo's are highly conserved in all land plants, with a calmodulin domain (calcium signalling related), also broadly transmembrane. There are 7 or so (opinions differ) clades of mlo based on their evolutionary history, the AtMLO11 (gene of interest for me) is from clade1 where it diversified very early on from other mlo clades 490Mil years ago, clade1 is related to morphogenesis (shape developement), very pretty pictures in this paper, the table one mentiones 4 papers about AtMLO11, will have to look into those, and that it has redundancy/syngerism with AtMLO4/14. It's expressed consitutively in leaf and root (thank god I am doing my cdna from leaf).
Yipee there is an entire section on AtMLO11/4 and what they do to roots, I am now starting to somewhat worry about my tDNA mutants because when I kept them on petri plates I don't remember seeing any funky root coling and I was supposed to see some, and cDNA checking pcr also shown only midly reduced expression (after 32 cycles but still there was sa lot of product)
I am passing out, and setting myself up for another 6h of sleep, backlog of work is growing.
It's very late, I wanted to rest a little before getting to work at home, and that got a little out of hand (4h of rest + an out of whack sleep schegule means that if I were to attempt to fix it I'd have to go to sleep 2.5h ago), I didn't manage to find the time to fill out the blanks in 01-12 (previous work) and 02-12 (last two weeks work) entries, I think it will take 4ish hours, maybe tommorow...
There is some downtime in the lab too I'd estimate 2h, when the PCRs are runing or gels are developing, maybe I should write on my laptop in the lab, I mostly just do my chinese characters anki (写汉字, HSK1 90% in deep green, baby!), and scroll social media.