Sample information

Methods

We used a DNA extraction protocol based on the insect adaptation of New England Biolabs’ Monarch Spin gDNA Extraction kit (Product # T3010). The specimen was incubated for 40minutes in a hot water bath at 56 degrees C.

Our first PCR reaction was set up on 10/14/2025 and:

• was a duplex reaction because we used both the Arthropod CO1 and Wolbachia 16S primers together.
• used an annealing temperature of   49  degrees Celsius.
• used this Taq polymerase: New England Biolabs OneTaq Hot Start Quick-Load 2X Master Mix with Standard Buffer (#M0488S
• Our first Gel image was taken on 10/22/25 and:
  • was run at 125volts for 24 minutes
  • used this DNA Ladder: New England Biolabs 1 kb Plus DNA Ladder for Safe Stains (product # N0559S)

• The second PCR reaction was set up on 10/29/25, used the same Taq polymerase as the first PCR reaction, and:
• was a single PCR reaction that used the Wolbachia 16S F and 16S R primer(s).

• used an annealing temperature of  55 degrees Celsius.
• Our second Gel image was taken on 11/5/2025 (date) and:
  • was run at 125 volts for 22 minutes
  • used this DNA Ladder: New England Biolabs 1 kb Plus DNA Ladder for Safe Stains (product # N0559S)

Results

Results for the 4 control lanes matched expectations after the duplex PCR because both positive arthropod and positive DNA controls had the 708bp and 438bp bands which represent the CO1 and 16sRNA genes respectively. The negative arthropod control had only the 708bp representing CO1 gene and the water also showed no signs of contamination, confidently confirming that the experimental results are valid.

After the single PCR for Wolbachia F and R primers, the confidence level lowered because the water control lane showed contamination.