Possible PCR Primers

I was able to narrow possible PCR primers down to about 3 pairs. It turns out, I do

not have to necessarily make my own primers, I can use primers that have already

been created to test them out. With this in mind, after researching universal primers,

I have decided to use a pair of PCR primers from last semester and 2 new ones. The

ones from last semester were 27F and 1492R. The primers I want to test are V3F,

V3R, V6F and V6R. Each primer has a specific sequence that will be replicated and

it can be seen in Figure 1. I found it interesting that the new primers have lowercase

letters in their sequence. After researching why this could be, thinking it could have

possibly been a typo, I found that lowercase letters can mean “low-complexity or

repetitive elements” (“Legend: Fasta”, n.d.).

Figure 1. The possible PCR primers that I plan to use during PCR. There are 6 in total, equaling 3 pairs. The sequences are listed next to each one starting from 5' and ending at 3'. 

Legend: Fasta. (n.d.). Retrieved March 30, 2018, from https://www.ncbi.nlm.nih.gov/SNP/snp_legend.cgi?legend=fasta

Chakravorty, S., Helb, D., Burday, M., Connell, N., & Alland, D. (2007). A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. Journal of Microbiological Methods, 69(2), 330-339. https://doi.org/10.1016/j.mimet.2007.02.005

PCR Primers

This week was quite slow because I have been researching possible PCR primers to
use for Polymerase Chain Reaction (PCR). The next step of my research is to
perform this but I cannot do so without these primers. With that said, I have to
develop my own primers from researching articles and protocols.. Even though I
am looking for universal primers, it is a little difficult to determine which one
would be best because certain ones work for certain bacteria or environments.
Since I am trying to identify unknown bacteria, I will most like have to develop
more than one primer. For example, one primer for gram positive bacteria and
another for gram negative bacteria. I will continue my research and hopefully
begin PCR next week!

Figure 1. The materials and process of Polymerase Chain Reaction (PCR). During PCR, the specific DNA that the primer targets is replicated over and over. If the primer cannot find that DNA, it means the sample does not have it and vis versa. ("PCR", n.d.).

PCR. (n.d.). Retrieved March 23, 2018, from
http://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-
and/pcr.html

Preparation of Research Poster

I started to prepare my poster for the upcoming research presentations. A few of them that I hope to present at will be the Estrella Mountain Conference, ASU Symposium and Arizona-Nevada Academy of Science Conference. I have already included items such as the hypothesis and abstract but still need to include my data. For instance, I have to provide a map that includes the locations each unknown sample was taken from (attached below). In addition, most of the bacteria from last semester’s were categorized in bacterial group numbers (attached below). I will make sure to upload my poster once I have it completed!

Figure 1. The 5 locations where creosote unknown samples from last semester's research project were obtained. The different colors match with the stars on the following maps and shows where it is. In addition to the location, the group number and locality is included. Rural (R) samples had to be further than 4 miles from populated areas and Urban (U) where within 4 miles of a populated area.  

Figure 2. The legend to the following maps that display the location of the creosote bush unknown samples. This also shows the total population per square mile by different shades of color.

Figure 3. This map displays the city of Phoenix. There are four unknown creosote samples that were obtained throughout this city. The samples, from left to right, were from White Tank Mountain, Phoenix College, Tempe and Coolidge. 

Figure 4. This map displays the city of Tucson. There is only one unknown creosote sample that was obtained from this city. The sample was from a suburban area in Tucson. 

Second DNA Extraction

This week, I performed the second DNA extraction on the unknown Larrea Tridentata bacteria. I was unsatisfied with the results I obtained a couple of weeks ago because the NanoDrop DNA values were low (well, at least to me). Due to these results, I thought I had done something wrong. With this in mind, I was hoping to get higher amounts of DNA during this extraction. However, that was not the case because I practically had about the same amount of nucleic acid. After this extraction, nothing changed and I will be using these samples for PCR. Below is the nucleic acid graph and data table for this second extraction (Group B).

Figure 1. The NanoDrop data collected from unknown bacteria in creosote bushes (group B). Five DNA extracted samples were used: A1, A8, A24, A26 and A27. Displayed with each bacteria is the amount of nucleic acid, 260 ratio, 280 ratio and260/280.

Figure 2. The NanoDrop graph from unknown bacteria in creosote bushes. The five displayed DNA extracted samples, A1B, A8B, A24B, A26B and A27B, are shown, along with the wavelength and the absorbance.