Dynamic Genome Lab Report

Tdghf Erg

Dr. Collin

11/22/2017

Dynamic Genome

Brown widow (Latrodectus geometricus) DNA extraction and primer design for sequence AcSp1CR to determine if a specific gene is present in the extracted spider DNA.

Background on spider silk and spider silk genes

The brown widow, latrodectus geometricus, was collected from San Diego, California (Vienneau-Hathaway, 2017) and is the extracted DNA used for this experiment. Most spiders use many different types of silks to perform multiple actions, for example the flagella form slik is used to create the capture spiral in a spider’s nest (Hayashi, 2010), while the piriform is used as a type of cement to attach their silk lines to a substrate (Hayashi, 2010). There are many other types of silk out

I amplified my extracted latrodectus geometricus DNA and performed PCR with pre-made primers to find the correct concentration of DNA to use for future PCRs. The samples used for this step were three tubes of varying DNA concentrations (1/10th, 1/100th, and 1/1000th), which were obtained via serial dilution of the original concentration. After this I designed primers for my C- Terminus sequence by choosing 350 base pairs upstream from the stop codon to avoid the repetitive region of my given sequence, AcSp1CR. By avoiding the repetitive region and only making primers for specific sections, we can avoid the repetitive region and therefore avoid having multiple bands in our PCR results. Once our primers were received, I converted the nanomolar concentration into microliters to get the amount resuspension buffer I needed to add to each, forward and reverse, primer. After that I set up a PCR to test whether my primers worked or not and determined that the concentration needed to be lowered using another serial dilution to get them to a lower concentration. Testing my new concentration of primers with my correct DNA concentration yielded an amplicon band the same as the expected product length of 500 base pairs. Now that both the DNA and primers were at the correct concentration all that was left was to run another PCR to not only to

geometricus gDNA was the 1/10x concentration. This result was obtained by following the parameters listed in table 1 and performing PCR for 20 minutes at 120 V. From there I designed primers for my specific sequence, AcSp1CR, by counting 350 base pairs upstream of the stop codon shown in figure 2.1. When I received my primers I needed to resuspend the forward primer and reverse primer with 238µl and 294µl respectively. Then I needed to test the working concentration for my sequenced primers as well using the parameters set by table 2. This yielded me the results shown in figure 2 where my primers did work, but had very intensely bright primer dimers that showed up in my results too. I diluted my primers using a 1/5x concentration instead of a 1x concentration, which using the dilution equation to get 20µl of primer with 80µl of ddH20, helped me make a new concentration of primer to use for my 2nd attempt. This attempt gave me the results shown in figure 2.2, where my primers again worked and this time with less bright primer dimers. This result also matched my expected product/amplicon length of 500 base pairs. Now that I know that my DNA concentration and primer concentration work for PCR I did another PCR to first of all, show that they worked, but second of all get my PCRed samples sequenced to determine if the gene I designed primers for is present in my actual amplicon. This PCR was suppose to