Since the last blog post and experiment, I have been focused on doing background research on how to design my own primers for use in amplifying DNA during the polymerase-chain reaction (PCR) process. Essentially, I am having to improve my knowledge of the structure of DNA and genetic sequences, so that I can determine which code sequences share similarities among the organisms I have selected for this study. Once I have identified a sequence that I find promising, I will have the primers made and proceed with that portion of the experiment.
In the meantime, I took the DNA samples which I have previously tested with my original study primers, and ran them through a PCR using Universal Rice primers (URPs). These kinds of primers are non-specific to any one gene sequence, and showed promise with bacterial DNA amplification in a study conducted by a prior S-STEM student.
The PCR I ran using the URPs was negative for strong DNA banding. Some DNA was present, but amplification appeared to be weak.This may be to one or a number of factors, as the variables in my experiment differed from the prior experiment in several ways. The DNA extraction itself was performed using a different protocol; the prior study used a commercially available kit, while mine used a simple SDS/alcohol method. PCR primer and DNA sample volumes also differed between the two tests. Furthermore, a major unknown when I attempted to compare the two studies was the primer concentration used by the other tester; that data has been lost to time, so I could not repeat those elements. Finally, I used an annealing temperature of 62.4°C during my PCR, as opposed to the 54°C used previously.
Next week, I will continue the background research portion of this project while attempting to replicate the previous student's experiment as closely as possible. Until then, happy science, and please enjoy this image from NASA of the Earth (taken from space.)
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