I really thought amplifying DNA would be easy. Molecular biology seemed simple enough in theory: extract, amplify, sequence, analyze. But I had no idea how difficult it would be to work with so many variables.
After my third failed polymerase chain reaction attempt, I started to wonder what I could be doing wrong.
Was I using the right concentrations of each reagent? Buffer at 1X, MgCl2 at 1.5mM, dNTPs at 200µM each?
Were the primers optimized for my DNA template? Had they been designed for the correct gene? Did they have the right length, melting temperatures, GC content?
Were the PCR cycles optimized? Was I using an annealing temperature within a few degrees of the primers’ melting temperatures? Were the denaturation and extension times long enough to assure proper replication of the DNA fragments?
Was the electrophoresis gel being prepared and run correctly? 1% agarose gel with GelRed in TAE buffer, run for about 10V per centimeter between the electrodes?
Or what if – shudder – the problem was in the DNA itself? What if the extraction had failed, and I’d have to start the entire project over from Square One?
No, no, calm down, I told myself. One step at a time. First, eliminate the variables you know aren’t the problem. There’s no reason the extraction should have failed; the quantification returned optimal concentrations of DNA. These are the same universal primers used in countless other studies, specifically designed for the cytochrome-b gene. The thermocycler and the electrophoresis apparatus have already worked before. There’s nothing wrong with the gel because the ladder and primers appear in the UV photos. That’s already five ticks off the checklist.
Now start with a positive control, a sample you know has worked with these primers before. PCR #4: did it fail to amplify along with the others? Yes. Good, then the problem isn’t the DNA.
Next step: new aliquots. Replace the water, the buffer, the MgCl2, the dNTPs, even the primers. Everything must be fresh to minimize contamination. Double-check the concentrations before preparing the mix. PCR #5: still no bands in the gel. No problem; just move on to the next step.
Try altering the reagent concentrations. Use more DNA template, double the primers, increase the amount of MgCl2. PCRs #6-8: nothing. Don’t panic, it’s all part of the process. You’re zeroing in on the problem now.
Adjust the PCR procedure. Increase the number of cycles, raise the annealing temperature in increments of 2ºC, lengthen the initial denaturation and final elongation steps. PCRs #9-12: still nothing. It’s okay, every failure is just another step closer to success. These tears are totally normal; nothing to be ashamed of.
For weeks, I tried everything I could think of. I replaced the reagent aliquots three times. I used half a dozen standard PCR procedures from various troubleshooting references. I ran gradient tests and touchdown tests and hot-start tests. Zero, nothing, zip.
Finally, after over a month of PCR attempts and no results to show for it, I was at my wit’s end. One morning, when I was alone in the lab, I broke down into a sobbing mess. I couldn’t look at the thermocycler anymore, that beast of a machine that I had to work with every day. I felt like a knight facing off against the same dragon over and over, knowing that hours of battling would only get me burnt every single time.
So I resolved to take a break from it all. No more PCRs for a while, not until I could work up the energy to start trying again. Today, I would just sit at the desk and read papers while indulging in a bag of cookies. Stale cookies. Really stale cookies. Seriously, how old were these?
I turned the bag over to check the date on the back…and that was when it hit me. Could it be? Immediately I dropped the bag and ran to the freezer. The stock solutions were stored in the back of the bottom shelf; I searched through them one by one until at last I found what I was looking for, printed clear as day inside the lid of the Taq DNA polymerase box. “Expiration date: Oct 2010.” Of course! No wonder none of my reactions were working: the enzyme was over five years old!
My first grin in weeks spread across my face, and I was suddenly overcome with an urge to shout “Eureka!” I hurried to my professor’s office and told him about the expired reagent. He quickly ordered a new stock and it arrived within two weeks. Sure enough, the next PCR I ran yielded the most beautiful set of bright DNA bands I had ever seen in a gel. I wanted to kiss that UV photo. My very first successful DNA amplification! And it only took two months longer than expected.
Never again will I underestimate the work of a scientist. Molecular biology may seem simple in theory, but having learned my lesson the hard way, next time I’ll be prepared for the challenge of working with too many variables.