Back to the drawing board!

As Thomas Edison once said, “I have not failed. I’ve just found 10,000 ways that won’t work.” Such is the act of science. It seems like for every ten times you try something eventually you will stumble across something that works. Unfortunately, I am still stumbling around in the dark. Progression is slow and the results I hope to see are even slower.  While this week as a whole was rather successful: the primers arrived, the mutagenesis kit arrived, media preparations are complete, the problem is the lack of positive results.

Using Stratagene’s kit, I ran a control PCR and a PCR with the five primers coding for amino acid changes in the pUCP28T plasmid background. Once the five-hour PCR was completed, the digest to remove any methylated (parent) DNA, and the new primer encoded plasmid was transformed into ultracompetent cells provided by the kit, the waiting game begins.

The “control” cells were plated on media that would induce a color change to indicate the mutations had been taken into the plasmid. The next day these plates showed a proficient number of colonies and an 85% mutagenesis efficiency rate. The plates on which the plasmid I want to induce mutations into did not show the same results. I plated the supposed mutated plasmid transformants onto trimethoprim (Tmp) plates (the plasmid that was transformed into the ultracompetent cells encodes for (Tmp) resistance. The plates were also grown at a lower temperature than normal due to the temperature sensitive phenotype being expressed. On all six of the plates with varied amounts of cell culture, none of the plates showed any sign of growth.

Back to the drawing board: One theory we have as to why nothing worked was the overwhelming abundance of primers we added. The next task will be to try with just one primer mutation to see if we can induce growth.

Edison also says, “Discontent is the first necessity of progress.” It is just a matter of time until there is a solution.



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