|Captive / Cultivated?||Wild-caught|
|Group||Wheeler High School|
12 ladybugs were collected from my room. They were found on the ceiling and shutters of my balcony doors. It was approximately 70° F inside the house. They were collected at 9pm on December 1st and immediately put in the freezer to preserve their DNA.
|Putative identification||Hexapoda Insecta Coleoptera Coccinellidae|
|Extraction kit||Edwards Buffer|
|DNA extraction location||Whole arthropod|
|Single or Duplex PCR||Duplex Reaction|
|Gel electrophoresis system||Standard electrophoresis system|
DNA extraction :
The 12 ladybugs were crushed with Cell Lysis Buffer then heated for 5 minutes. Next they were vortexed and centrifuged, creating pellet debris. The supernatant was extracted and mixed with cold isopropyl. This mixture was mixed and centrifuged to pellet genomic DNA. The supernatants were extracted and ethanol was added to the pellets to wash the DNA. This was centrifuged and the supernatants were extracted again. Te buffer was added to each of the 12 tubes.
A PCR cocktail was created with Arthropod_F primer, Arthropod_R primer, Wolbachia_F primer, Wolbachia_R primer, water, and Taq Master Mix. This cocktail was combined with the DNA from each of the 12 ladybug and placed in a thermal cycler.
Gel Electrophoresis :
The DNA was run in gel electrophoresis and results were obtained.
About 5 ladybugs had Wolbachia present. These were in lanes 4, 6, 7, 9, and 10. There were clearly two bands while the rest had either one band or were unclear. The positive control , negative control , and Wolbachia DNA did not show up in the gel electrophoresis.
|Explanation of confidence level||
Though some bands were smeared, 5 arthropods were infected with Wolbachia. These 5 were extremely clear and distinguishable. Most of the other 7 had one band visible. The three controls were tested and validated through a different gel.
|Wolbachia 16S sequence||
|Arthropod COI sequence||
|Summary||The Coccinellidae was found to be postive for Wolbachia.|