Between Here and the Sky- Chapter 2: Primers

As he neared the end of his candidature the amount of work still needing to be done loomed in front of Marshall like an angry hydra- each head a different branch of the project that somehow had to be coaxed and cajoled into a smooth and consistent thesis. Time was running out. The days were lengthening with the coming of summer and with a determination brought about by the fear of failure he forced his mind to burn from sunrise to sunset.

By now he was used being the first in the lab. Well, it was either he or his lab mate Yoshi. They had started their PhD’s at more-or-less the same time, albeit from different directions, and had worked together quite a lot, helping out with the more time consuming and repetitive elements of their experiments. Both thought it was a good thing to have some companionship through the ups and downs of their candidature, even if working that closely with another person for so long inevitably led to squabbling, bickering, the odd fight. In effect they were like brothers forced together by fate or whatever grand design there is in the world, if indeed there is any.

Marshall threw the switches to the fluorescent lights to revel the usual white starkness of the laboratory. Bottles of clear liquids labelled with autoclave tape lined the shelves; the air-conditioning sprang to life with the lights. Overnight the lab had developed the fetid stench of mouse shit and piss, a consequence of the renovations being done in the animal house below disrupting the extraction and conditioning of the surrounding rooms. Marshall screwed his nose up in disgust. He knew he would take half an hour before the smell would clear. In the meantime he would have to suck it up.

He slipped the starched lab coat over his shoulders and pressed the studs closed at the front before slouching around the bench. In a well-practised motion he ferreted out some gloves- large- from their cardboard box. He cursed as the latex band around the wrist tore and he had to put on another. Once his hands were secured and the polystyrene box filled with crushed ice Marshall delved into the freezer to collect his reagents. The laminar flow hood started its familiar rumble as he turned it on and leaned its metal sash against the bench. The radio noise was lost in the din. Marshall grabbed a plastic bottle and sprayed ethanol over the metallic interior of the hood and wiped it down with a paper towel. He placed the frozen tubes of reagents to thaw on the grill over the rush of filtered air. He stuck a yellow post-it to the glass panel separating him from the hood and placed 72 tiny plastic tubes into their holes in the frozen metal rack and selected a yellow-tip pipette. Following the recipe he’d scrawled onto the post-it he set to work.

16 samples, duplicate; 4 standards;  p53 and GAPDH ((16x2)+4)x2
	1x	36x (+8%)
Sybr	5	194.4
Primer-F	1	38.88
Primer-R	1	38.88
H2O	2	77.76
cDNA	1

Without really thinking about it, Marshall aliquoted nine microlitres of the cocktail into each of the tiny tubes. He reflected with wonder at the number of such pipette motions he’d done over the course of his PhD, and how many more he would do before he finished. Millions. A new robot had recently been installed in the department to do such tasks for them, but both Marshall and Yoshi preferred the absolute control of doing the entire procedure themselves. Now that they were nearing the end neither wanted to start messing with a system that they both knew worked. Of course, if something went wrong with their system there was no one to blame but themselves, but to their minds this was a small price to pay for absolute confidence in their results. Marshall sighed and kept pipetting as cramp started to set in to thumb and wrist.

Yoshi walked through the doorway as though searching for something. Marshall looked in his direction and Yoshi idly waved and entered the cool room. A moment later he stepped out and called across the room.

“I’m gonna run a gel. Do you have any samples you want run?”

Marshall pondered at the ceiling a while. “Nah.”

“I’ve only doing two samples. It seems such a waste, but it beats trying to write.”

Marshall laughed, then as Yoshi turned away, returned to work. He’d lost his place. Lifting tubes out of their slots he found where he was up to and continued in his precise but absent-minded way. Once each tube had been filled he turned on the next hood, removing its sash in one practised movement and transferred the rack into it. He lined up the 16 experimental samples and the 8 standards in order on the grill to thaw and, taking in a deep breath started add the cDNA in careful sequence. He did everything in duplicate to control for any error in his pipetting. All checks and balances must be in place and accounted for. He had to be meticulous. Despite having done this procedure a hundred times, he still had to stay focussed, particularly on this step. Any slip up- putting the cDNA into the wrong tube, forgetting to change tips between each action, losing his place- would mean he would have to start the entire process all over again. He held his breath involuntarily with each motion of his arm and thumb.

He capped the tubes and moved everything into the PCR room, sterilising and shutting down the hoods as he did so. As he turned his back a noise like a gunshot ricocheted off the walls, splinters of plastic thwacked off his jeans. Marshall flinched and turned to be struck in the face by a pair of gloves. Yoshi appeared in the space vacated by the falling gloves.

“Jesus! Motherfucker!”

Yoshi leant against the bench, pointing and laughing.

“You’re a pest.”

“Yep. What are you in so early for?”

“To be hassled by you, whadya think? PCR.”

“Yeah? What are you doing?”

 “p53.” Marshall’s face fell into a pout.

“Haha! p-fucking-53. Sucks to be you.”

 “Tell me about it.” Marshall grimaced. It had been an ongoing pact between the two of them that they would never look at p53. Papers were coming out all the time implicating it in this disease or that pathway, but to their minds it was mostly a load of hot air. They refused to believe that life hinged on one single gene, that if that one gene were to go bung then the whole system would collapse. To them it defied the fundamentals of evolution. Instead they preferred to toil away within their own little obscure niche trying to make a contribution to science without getting caught up in the flashing lights of scientific fads.

“What the hell are you doing that for?”

“I’m just making sure it’s not altered after knockdown of PGC-1α. Just validation, really.”

“You realise you’ll have to dive down that rabbit hole for your lit review, you know.”

“I know...” Marshall looked downtrodden. “Any better ideas?”

“Pfff. You’re on your own. I’m not touching that. Let’s just hope for your sake it’s not changed.” He laughed and Marshall grinned wryly. If it turned out badly he could just write it off as outside the scope of the study and not include it in his thesis. Just a mild case of fraud by omission, really. He turned back towards the PCR machine and loaded the samples into the rotor, being careful to place them in their specified order, and started the run.

95°C for 5 minutes
60°C for 5 seconds
72°C for 5 seconds	-- 45 cycles
95°C for 1 second
55°C à 95°C (0.5°C stepwise)

It would take just over an hour.

Satisfied that the machine could take care of things without his supervision Marshall returned all his reagents and samples to their respective boxes in the freezer, disposed of his gloves and coat and returned to his office. As he switched his computer on he grabbed his jar of coffee and heaped 2 spoonfuls into his oversized mug. Before trotting downstairs he logged on to his computer so it would be primed for his return.

            The screen was brightly lit with a photograph from the farm, but the laptop was still chugging away loading all the settings. “Fucking Vista,” he muttered under his breath. He clicked on the taskbar icon for the internet and the page loaded to the image of three elderly and very naked men in the act of performing an array of sexual acts with each other. “YOSHI!” he yelled as he jabbed at the esc button at the top left of the keyboard.

Giggling could be heard down the corridor. All of the occupants of the third floor were meticulously vigilant about locking their computers when Yoshi was in the vicinity, but for whatever reason- he blamed it on the early hour and lack of caffeine- Marshall had failed to follow protocol. He withdrew his ruler and a handful of elastic bands from his drawer and stuffed a couple of stress balls down his pants to ferment amongst his junk and, locking his computer, set forth to hunt.

It hadn’t been the best of days. To start with it was a Friday, and really nothing potentially momentous should be started on a Friday. That was his first mistake. The second was to take a shortcut. Rather than first testing and optimising the primers and experimental conditions Marshall had skipped ahead and gone straight to running the unvalidated primers directly on his samples. If it had worked it would have saved maybe a weeks work. But as it stood he had instead wasted time, not to mention effort, his precious samples, and the hundreds of dollars in costs. He couldn’t get them back now. Having looked at the computer read-out 40 minutes after it started he’d marched straight back to his desk, unlocked his computer, and sent an email around to his usual accomplices stating that he was heading to the pub early and wasn’t planning on leaving without a security escort at closing time. Until then he had 4 hours to kill.

            He did the usual whip-around of the major news sites to keep up to speed with the world, and tried to at least be productive in his time wasting by cataloguing all the chemicals and machinery he’d used during his project for his Methods chapter, but had instead become caught up chatting with anyone he ran into, anyone that would stop and talk to him of their weekend plans and dreams. He even cornered the head of department to discuss the weekends football tipping and to taunt him over his team’s poor league position. What should have been a half hour task was stretched out to an hour and a half. Impressed with his efforts he rewarded himself with an early lunch.

            He rustled up a group of like-minded postgrads and headed down to Broadway to grab takeaways to eat on the departmental balcony overlooking the med students on the grass below. They sat and ate and shot idle thoughts into the wind for what didn’t seem long enough, until their sense of duty and habit gnawed at their conscience and they burst apart to their own desks or benches within the corridors of the building. Behind their eyes was the Tav later in the afternoon, when all of the week’s troubles and disappointments could be put aside as they toasted each other, the weekend, the world, but until then there was work to be done.

A pile of journal papers was fanned out over Marshall’s keyboard when he returned, evidence of supervisor surveillance. He blew out his cheeks and quickly skimmed the titles- p53 presented a common theme. Fuck. An early finish was wiped from the table.

He unlocked his computer and checked the availability of the PCR machines. His favourite was booked up for the afternoon, but there was a 2-hour slot available on the cantankerous old beast. He shrugged. He was only running optimisation. The beast would suffice. He wrote in his booking, and set about drafting the conditions and settings he’d trial.

“What’s up?” Piers walked into the room through the door at the other end of the office. He wiped at a lock of black hair that had shaken loose and fallen over an eye.

“I’ve just created a whole heap more work for myself.”

“Fuck.”

“Have you knocked off for the day?”

“Nah. Not yet. I’m delivering some documents to the Dean’s office.”

“Isn’t that up your end of campus?”

“Yep.” Piers grinned like a child knowingly doing wrong. “I’ve got a simulation running anyway. Nothing much else for me to do. Just wait.” He sat and spun around on a chair with his feet tucked in to his chest. Marshall watched idly. Piers stopped, facing his friend. “You guys going to the Tav later?”

“Planning on it. I need a fucking drink.”

Piers laughed. “That’s not all you need. Alby’s band is playing at the Cellar tonight. Got their album launch. Wanna come?”

“Sure.” He thought for a moment into dead air space. “Reckon you could get us on the door? I’m running a bit low on cash.”

“I can see what I can do. I’m on the list. Don’t know how many other spots he’s got free.”

“Cheers.”

“They’ve improved a hell of a lot in the past couple of months. Some of their new stuff is fucking immense.”

“Yeah?”

“Yeah. Apparently they’ve got some record labels sniffing around. Like, big ones.”

“Shit, good on ‘em.”

“He’s pretty exciting. And you know how excitable he can be.”

Marshall laughed. He’d met Piers in their first year Chemistry labs, and had straight away had clung to him. He’d only just moved to the city and aside from a few schoolmates in other courses he knew no one. He had to grab hold of whoever would tolerate him. Luckily, Piers had turned out to be just the type of person Marshall had been looking for, and they quickly became close friends.

Whenever he’d gone around to Piers’ house, his younger brother Alby would be hanging around wanting to get involved in whatever they were up to. He could get enthusiastic about anything; even the act of sharing a joint around the table on the patio would impart a wide-mouthed grin on Alby’s face. He always seemed to be excited about everything. He certainly wasn’t like all those 16 year olds he had seen loitering around Forrest Place- all frowns, attitude and eyeliner.

“Have you caught up with that girl from Tuesday night?”

Piers smiled sardonically. “Nah. I messaged, but she didn’t message back. I guess I’ll leave it as a once off.”

“Better than nothing I s’pose.”

“I guess. Well there’ll no doubt be some girls there tonight, and you never know your luck- Alby’s friends are all a little mad.”

“Mad and single?”

“Dunno. Probably?”

“Well I guess I’ll come then. Let me know if you can get more comps.” Marshall turned back to his pencil and paper. “Now if you don’t mind, I have to get some calculations done, and then there are some Flash games that won’t play themselves.”

Piers stood and slapped him on the back. “I’ll be back at 3:30 to collect you for the Tav.”

“No worries. I’ll let you know if we go earlier.”

Piers wandered out the door and down the corridor to Yoshi’s office. Marshall pushed the buds into his ears.