This last week was probably the most stressful of the term. And also I still don’t know when my last day will be.
So our research project was due last Thursday at 5. Unfortunately, we did a lot of last minute things and things didn’t go as smoothly as expected. Fortunately, most things were fixed before we handed it in, but still -_-
We’re learning a bit about epigenomics now, and I understand the biology part but not so much the bioinformatics side of things (this goes for many things in this course) and it’s also explained later in the lecture…
I wonder if we have tutorial next Friday lol cuz it’s the last week. Or better yet perhaps, how many people will show up. Wow I can’t believe it’s the last week already. I am glad I only have one exam.
Finished making dat library. The material I was using was genomic DNA from a single cell that had undergone whole genome amplification (WGA), specifically something called multiple displacement amplification (MDA). Basically, MDA allows for amplification of a small starting amount of DNA. It is advantageous to the polymerase chain reaction (PCR) because it creates longer fragments and supposedly lower error frequency. I think it works by using random 6-mers (ALSO KNOWN AS HEXAMERS OK TYLER) as primers. Also the reaction is done at room temp.
Random primers are used to minimize the bias in amplification (although obviously it doesn’t eliminate it). Unfortunately, due to the use of random primers which can theoretically amplify almost any sequence that is long enough, this means that any contaminant DNA would also be amplified lol. And of course, polymerases (or any enzyme purified from a cellular system) have plenty of DNA contamination.
I wonder why they (companies that sell enzymes) don’t add DNases to polymerase mixes for a while and then heat them to inactivate the DNases (assuming that the polymerases won’t denature, and if they’re for PCR then they really shouldn’t).
Anyway, so yeah since single cell sequencing is subject to a lot of sequencing bias (and loss of material during library construction), whole genome amplification techniques such as MDA are used before any library preparation in order to generate more material.
On Friday I carried out a qPCR. We set up our qPCR in 384 well plates. qPCR set up is quite cumbersome and probably one of the things that stresses me out the most. The viscous brew, the fact that I have to pipet such small volumes and everything has to be in triplicate… and I don’t like pipetting in 384 well plates b/c the wells so small ):