From March to July, my major sequencing problem was that I would take perfectly good amplified DNA, run it through the sequencing process and ethanol precipitation, put it in the machine, and would come out with largely nothing...it was as if the wells were just blank, and my results were worse every time. It took me until the end of July to find out (and test, and prove) that my problem was bleach. Although the rubber septa that fits over the plastic plate is meant to be reused, the plastic plate is not...and when I reused it and rinsed it out, I was adding bleach to kill any remaining DNA so it wouldn't confound my results. Turns out the bleach lingered and killed new DNA too.
But from the end of July up until now, I was still having problems. I was finally getting results, but they were so crappy (short sequences with lots of clutter and very low signal strength) as to be totally unusable. I tried different PCR parameters. I did primer research. I had umpteen meetings with my lab manager and other grad students. I changed my reagents. No luck.
I was running out of time. I need to make some serious headway on this project before my advisor kills me, and I'm taking a class right now where the only two assignments (one due in a month) require some analysis of our DNA sequences, of which I had almost none. It was like a clog in the drain, where the sink was filling up with more and more samples (about 150 in all) and none able to get past that final, crucial step. Plus, I had contamination problems to sort out on at least two separate occasions (problems with the length of my pipet tip), and of course, it affected one of the most expensive substances I use (Taq).
I've spent the last month slowly falling apart. I'd get up and go to work in a daze. I'd burst out crying in my office, in my lab, in my car. I started to get panic attacks and hyperventilate, which has never happened to me before. I started dreading my email inbox, afraid of getting word fron Steve that I was canned. I would take circuitous routes back to my car at night to reduce the risk that he'd see me leaving the lab. I would arrive at work at 8 AM, leave at 6 PM, Mondays through Saturdays. I started speaking in a monotone. I went home for PAX and freaked out my first night home, thinking that Steve would find out I was not in the lab (and avoided all cameras at the event, just in case). I considered going to the doctor for anti-anxiety meds, just so I could think straight.
Being such a good student for so long has denied me the ability to properly cope with failure. This was difficult the first time I had sequencing problems, but after the 5th time, the 10th time, the 40th time...I was starting to go a little crazy. The only part of my day I looked forward to was going to sleep at night.
I haven't been able to write any of this down for months.
Well, today, I think I've proof that I solved the problem. After Cort tested some of my sequences a few weeks ago and got good results, we figured it had to be a technique thing. Thus followed a two-day session of me going through my entire sequencing procedure with Cort sitting there, watching my every move. He made a few suggestions that helped speed and economy, but the suggestion that ended up making the difference involved my very last step, which is rehydration.
After washing my DNA several times and centrifuging it, there's a point where the DNA is in a tiny pellet at the bottom of a tube and allowed to dry out. After drying (to get rid of the ethanol), I had to pipet 10 microliters of formamide into each tube, pipeting up and down a few times to "wash" the sides of the tube and be sure I got everything. Which is what I did. Turns out, "washing" has to be much more rigorous...trickling the liquid down all sides of the tube as you rotate, spending about two minutes on each tube instead of about 20 seconds. Nobody told me this. It wasn't in the directions. It's just one of the things you're "supposed to know," I guess.
Between that and trying to add a tiny bit more DNA to each tube from my exoSAP reaction, I think I got it. I spent about an hour "washing" my tubes last night (and slowly going blind...10 microliters is not a lot of liquid). I put the samples in the sequencer this morning, and 15 of my 16 samples came out completely fabulous.
I'm not ecstatic. I've been too depressed for too long to bounce back that quickly, to say nothing of the work I have left to do. But it's taken a weight off my chest. And when my chest remembers how to breathe again, I think I'll feel better.