The internet refers to my situation as the “Pilot error hypothesis”; I call it sabotage. When a young scientist initially begins doing PCR on their own, it can often go wrong. This hypothesis states that it is a common issue simply due to inexperience. The first two weeks of PCR were excellent (Under lab-mate Yanan’s supervision of course), it was looking like I could finish my project on time. However, the past week has surely put me behind. For a reason unknown to me, my DNA never comes out on my PCR.
This is an issue. I have tried: changing the amount of DNA in the initial mixture, creating a new DNA template, checking DNA concentrations in the template, changing the PCR program temperature, using brand new buffers, new primers, new polymerase, changing PCR machines, changing electrophoresis machines, increasing/decreasing electrophoresis voltage, everything I can possibly think of… but nothing has given me the correct results.
It takes nearly the entire day to learn the results; there is no DNA in my agarose gel. Then I do it again. I have been working 10-6, a couple times coming back at 8:30 pm. It is really frustrating to work all day, and then finish with no results.
Last Thursday was even more frustrating. At 2 pm, after waiting all day for the PCR to finish, I found the machine had been turned off! As if my week was not discouraging enough, someone ruined my samples! I had to completely re-start the day’s work.
Today, I booked the PCR machine in advance. (THE PROPER THING TO DO) Then, when I went back downstairs with my samples, someone had put their samples in the machine and had started running their PCR! This is a 2.5 hour procedure, so obviously I was annoyed.
Having 10 failed attempts at PCR is clearly upsetting. Today, (otherwise known as attempt #11) I am trying to use a new gene, with a new primer. If this does not work, then I will have to think of a new method for #12… While reading a National Geographic (I am finding myself with a lot of free time while PCRs are running…) I read the phrase “Persistence commands success”. Generally I do not give inspirational quotes a second glance, however I find this exceptionally relevant. I kept it in the back of my mind for the past 5 or so PCR/electrophoresis attempts, and I think it is an appropriate summation of the past week.
Hopefully next time I report back it will be with positive news!