Post 3: PCR (Problems Continue in Research…)

Hello All,

 As you may have guessed from the title of this post, sometimes research does not go as planned or as expected. We have recently begun Polymerase Chain Reaction (PCR) work as a part of our analysis of the community profile of the bacterial population in Lake Matoaka and it’s watershed. Here our research hit a snag or two. PCR was not working as expected and our research was stalled. Our focus shifted from community profiling to optimizing PCR.

Briefly, PCR is a means of amplifying small quantities of DNA. Using an enzyme called Taq Polymerase, the three step process amplifies DNA over a period of several hours. The first step denatures the double-stranded DNA, making it single-stranded. Without single stranded DNA, amplification is not possible. The next step is the annealing step. The primer needed for elongation is annealed to the single-stranded DNA. The third and final step is the elongation phase. Taq Polymerase adds complimentary nucleotides making a new, semi-conserved strand of double-stranded DNA. This entire process takes a couple of hours, the results of which are assessed by gel electrophoresis. For more detailed information about PCR please see, http://www.dnalc.org/ddnalc/resources/pcr.html.

This is a fairly straightforward procedure that can come with a plethora of problems. After running several gels of different sets of PCR products, we had nothing to show for it, but pictures of empty gel lanes. It was very frustrating. Troubleshooting PCR began with literature searches and contacting the manufacturers of the various elements of the process. One solution kept popping up, Bovine Serum Albumin (BSA). BSA is used to prevent non-specific binding of the Taq Polymerase to plastics. As the tubes we use are plastic, the BSA coats the sides of the tubes and prevents the Taq from biding to the sides of the tubes. The Taq is now free to bind to the DNA in the tubes.

We have not had perfect success with PCR every single time. We joke that there is a certain amount of voodoo involved in getting PCR to work. Aside from taking extra precautions in using aseptic technique, there is little else we can do.

All in all, we have each learned a valuable lesson. Research is about solving problems and sometimes those problems arise in the methodology. Frustration and failure are innate facets of research. It is better that I understand that now  in dealing with something as simple as PCR. My chosen career will not be easy. Like I said in the title, problems continue in research and that is why they call it research!

 Dana