Sample information

• Next to a garden
• Outside open area
• Crawling out from underground

Methods

1. We used a DNA extraction protocol based on the insect adaptation of New England Biolabs’ Monarch Spin gDNA Extraction kit (Product #T3010)

2. The specimen was incubated for 30 minutes in a hot water bath at 56 degrees C.

3. Our first PCR reaction was set up on October 14th,2025 and was a duplex reaction because we used both the Arthropod CO1 and Wolbachia 16s primers together

4. used an annealing temperature of 49 degrees Celsius

5. used this Taq polymerase: New England Biolabs OneTaq Hot Start Quick-Load 2X Master Mix with Standard Buffer (#M0488S)

6. differed from the written protocol (time differences, accidental mistakes, etc.)

7. Our first Gel Image was taken on 10/21/25 – was run at 125 volts for 20 minutes

– used this DNA ladder: New England Biolabs 1kb Plus DNA Ladder for Safe Stains (product #N0559S)

• Our second PCR reaction was set up on Oct. 28, 2025, used the same Taq polymerase as the first PCR reaction, and:
• was a dublex reaction that used the arthropod F and R and Wolbachia R and F primers.
• used an annealing temperature of 49 degrees Celsius
• differed from the standard protocol (differences in amount of DNA per sample, accidental mistakes, etc.) in that: – used 1.5 UL of (+) DNA control with .5 UL

Results