draft tai

March 30, 2022

Sample information
Picture
Photos by: TD
Location
Collection date 09/14/2022
Captive / Cultivated? Wild-caught
Group Woburn Memorial High School
Observations

The pill bug was caught by someone else, but I had the chance to use the pill bug for the Wolbachia experiment. The pill bug caught is an isopod that is grey and very tiny.
Putative identification Arthropoda Crustacea Malacostraca Isopoda Armadillidae

Methods
Extraction kit Edwards Buffer
DNA extraction location Partial abdomen
Single or Duplex PCR Duplex Reaction
Gel electrophoresis system MiniOne
Buffer TBE
DNA stain GelGreen
Gel images
Protocol notes

DNA Extraction protocol

Results
Wolbachia presence Yes
Confidence level High
Explanation of confidence level
The purpose of this experiment is to isolate DNA from organisms and determine if there is Wolbachia DNA present by using PCR. Arthropod samples were lysed and the DNA was extracted from the samples. PCR was used to amplify the CO1 gene found in arthropods and the W16S gene found in Wolbachia. The CO1 gene has a length of 708 base pairs and the W16S gene has a length of 438 base pairs. There were 5 samples that were run through PCR: a sample, +Wolbachia, -Wolbachia, a +DNA control, and a -DNA control (water).  The +Wolbachia and the +DNA control were expected to have two bands, one at 708 BP (CO1) and one at 438 BP (W16S). As seen in Figure 2, well 4 (+Wolbachia) and well 6 (+DNA control) had two bands as expected. The -Wolbachia sample was expected to have one band at 708 bp. As seen in Figure 2, well 5 (-Wolbachia) had one band as expected. The -DNA control (water) was expected to have no bands as it should not contain DNA. As seen in Figure 2, well 7 (-DNA control) did not have a band. Lastly, the sample should have either one or two bands, depending on if the arthropod was infected. As seen in Figure 2, well 3 (sample) had one band at 708 bp and no band at 438 bp, meaning that the sample was not infected with Wolbachia. Due to the controls having the expected results, the experimental results are valid. The pill bug is confirmed to be infected with Wolbachia.
Wolbachia 16S sequence
Primers provided by Wolbachia Project
BLAST at The Wolbachia Project   BLAST at NCBI
Arthropod COI sequence
Primers provided by Wolbachia Project
BLAST at The Wolbachia Project   BLAST at NCBI
Summary The Armadillidae was found to be postive for Wolbachia.
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