PCR and sequencing HMY7

Hello readers! (of which I am sure there are many)

Well, I have reached the end of the second week of summer research. So far, I have been working on the project that I have been on since the Spring semester. It isn’t my allantoin pathway work yet, but it will lead into that project, so I am excited to finish up this stuff so I can look into the allantoin research. I have been doing PCR, PCR, and some more PCR! I did PCR for all of the genes previously found to be significant to plastic adherence for the strain HMY7. In the Spring, I worked on the genes in the strain HMY355. PCR proved…difficult. Many of the genes in these two strains are particular to say the least. So, I ended up with a bunch more PCR hours under my belt, but only about 50% of them worked. But! That’s okay. I pushed on and did PCR clean-up for the genes and the corresponding segregants (high and low plastic adherence segregants) that did work. I then did a big push for sequencing and just submitted the samples. Hopefully the sequences will be back in a few days and I can begin analysis! What I will be looking for is a systematic difference (an SNP or change in allele) between the high and low segregants because this means that that difference is probably crucial to determining whether or not the segregant will have high or low adherence.

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Milkweed Connectedness Update 1:

My original plan was to use common Milkweed as a study system to understand the impact of clonality and group survival. By intentionally adding a pathogen, such as an herbicide, the spread of the negative effects can be witnessed in a clonally connected plant. The goal of this experiment is to see how far the pathogens travel in a patch, how long it takes for other plants to die, and if there is any preferential sharing. For instance, sometimes younger plants are favored in sharing. Herbicide will be used as a proxy for connectedness and physiological integration. 

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Conclusion

The goal for this summer was to have sampling, DNA extraction, Polymerase Chain Reaction (PCR), and fragment analysis sent off (to be later analyzed) completed on all 400 of the subsamples. Sampling and DNA extraction were finished. PCR unfortunately was not, delaying fragment analysis as well. Though the PCR protocol was developed during spring semester of 2017, new primers and a host of other problems made this step tricky. The first batch sent off for analysis came back blank, requiring adjusting of the protocol and the second had nebulous results. A delay in shipment meant that only 3 of the 7 primers were available for much of the summer. However, all extractions were completed successfully, and in the last remaining weeks of July, PCR was done on all 400 of the plants for 3 of the primers. All PCR products were shipped off for fragment analysis. Training began on using a fragment analysis software, so once results do come back, analysis will be smoother.

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Labwork (PCR)

Following DNA extractions, it was time for amplification of DNA through Polymerase Chain Reaction (PCR). PCR amplifies the DNA by denaturing, copying, and synthesizing over and over again. Denaturing occurs via temperature, first during the DNA extraction process, and then again using the Thermocline machine (or PCR machine). The thermocline helps to regulate temperature allowing for PCR to go through its different steps, such as synthesis or denaturing. Synthesis occurs using a special polymerase Taq.

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Labwork

Following the collection of data and samples, it was time for lab work to begin. The first step of my procedure was DNA extractions. With around 800 plant samples, I quickly realized financially and time wise doing all 800 would not be practical. This led to many discussions on how to subsample. Was it better to do more transects, with fewer plants from each transect? More plants, with fewer coverage of overall transects? How do you account for the difference in densities between transects? Overtime, with more and more discussions, it became clear that sampling more transects would be a better option, even if that meant fewer plants per transect. Additionally, for any transect with ~30 or fewer plants, the entire transect would be sampled. For any plant over that, a subsample would be done using a random number generator to randomly select which plants should be extracted.

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