FUCK! I’ve had it with this goddamn fucking virus! WHAT THE FUCK DO YOU WANT ME TO DO! I’ve swaddled you in your favorite media, I’ve varied the salt concentration, I’ve increased the incubation times. Fuck, I’ve done everything but inserting you up my ass via an enema! Why in the name of all that is holy, unholy and slightly tarnished by rum WON"T YOU FUCKING GROW!
Ok, this is virology geek time: Be warned.
I’m working with a strain of HIV – NL4-3. No big whoop. We got it from the NIH’s HIV reagents. Ya know where scientists send in samples that get cataloged, expanded and distributed to other researchers across the nation?
So, someone isolates the NL4-3 genome. Yay, that’s a hard days work. Then they cloned it into a plasmid (circularized piece of DNA). From the plasmid you can either insert it into a bacteria or into a mamalian cell. If you put it in the bacteria, each time the bacteria divides it will copy the plasmid DNA and each daughter bacteria cell will have a copy. From here you can grow up a few million bacteria then you can use a plasmid extraction kit and kill the bacteria and isolate the plasmid.
If you stick the plasmid into the mamalian cells then you’ll start growing virus.
Now, to grow up the virus itself, you first NEED to have enough plasmid to transfect the goddamn cells! I"M OUT! I’ve BEEN out for months now! Luckilly I made up enough virus previously I still have my stocks of the virus. BUT NOOOOO! Now I need to grow FRESH virus under a variety of conditions to see what affects pathogenesity! So I’m FUCKED I need more plasmid!
So, I take a small amount (3 nanograms) of DNA and electroporate some JM109 cells (bacteria cells) to get the plasmid into a bacteria cell then I plate those transformed bacteria onto a selective agar that only allows those bacteria with the plasmid in them to grow.
After letting that grow up for 1 day, I seed 1 single colony (you pick one colony because each colony came from a SINGLE bacteria–this way you are ensuring that all bacteria in the colony are clones of each other) into some LB-AMP (It’s basically beef extract + yeast extract + salt + antibiotic selection). I then grow that over night and then do the plasmid isolation and prep.
Doesn’t fucking work.
I try AGAIN.
Doesn’t fucking work.
I vary the incubation times.
Doesn’t FUCKING work.
I vary the salt concentration and pH.
Guess what! IT DOESN"T FUCKING WORK!
I’m now totally out of NL4-3. I now have to go into the our MASTER stock of the fucking plasmid.
In use 5 TIMES more DNA for the transformation.
Still, won’t work.
I go talk to my boss.
“Use the glycerol stock”
(Glycerol stocks-- Are bacteria that ALREADY have the plasmid inserted into them that are then frozen down in a 10% glycerol solution at -80C. Having these effectively cuts out the first day of the experiment. Since you already have the bacteria w/ the plasmid you don’t have to do the electroporation.)
So, I use the glycerol stock, which after checking is DIRECTLY from the NIH’s stocks.
Doesn’t FUCKING work.
“Try again, let it grow longer”
I GOT DNA! Not much dna. I ran 2 250mL cultures. Generally that’s enough for 600-1000 ug of DNA.
I got 106.3 ug.
That’s TERRIBLE.
So I go back and talk to my boss again.
After a few days guess what I learned!
“The NL4-3 plasmid doens’t like JM109 cells!” Which means the plasmid doesn’t replicate well, which means the bacteria doesn’t grow much, which means I get shitty yields with bad DNA.
Which means, the assnugget who stuck the NL4-3 plasmid into the JM109 cells for the glycerol stock FUCKED ME!
I’ve got a plasmid I NEED badly in the wrong fucking host cells! This means that it won’t grow up in any meaningful concentrations, that it yields damaged/incomplete DNA half the time I.
I’ve run this experiment 2 more times (brings me up to 11 times total) since saturday. AND NEITHER WORKED! This time I didn’t even get 1 mg!!! of bacteria to grow after 20+ hrs in the shaking incubator!
I’m sick and tired of my boss asking me for data I can’t BEGIN to generate cause of this FUCKING plasmid!
When I figure out who the Nobel winner who thought up the brilliant idea of sticking this plasmid in a line of bacteria that WON"T GROW IT I’m going to take his glycerol stock, get some on an innoculation hoop and INNOCULATE HIS RECTUM 25 FEET UP HIS INTESTINES!
I bet the fucking bacteria will grow better there! It’s ecoli after all!
Because of this twit, I’ve been making GALLONS of LB broth for growing bacteria.
Ever made LB? It’s FUCKING NASTY!
5g of Yeast Extract. A finely milled powder, so when you open the container it gets EVERYWHERE. And yes, it smells like yeast left to rot. It TASTES like rotting bread. ALL my clothes smell like it. My car keys smell like it. My ID badge smells like it!
10 g of NaCl – no biggy
10 g of Bactotrypone – Holy fucking shit.
It’s powdered COW! It looks like someone took a cow, and rendered it down to a powder. When you taste it, and you WILL taste it when you open the container. This thick, finely milled powder flys everywhere. And when you breathe in, it tastes like STEAK!
Which is FUCKING WRONG!
ARRRRRG.
So now I’m back up shit creek. The unfortunate thing was, if BOTH experiments that I used the plasmid up in had worked I wouldn’t have to grow more.