Sample information

When I collected the bug it was stuck in a spider web, however it was still alive.

Methods

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

2) The specimen was incubated for 40 minutes in hot water bath at 56 degrees C.

3) Details that differed from the written protocol were the change in pipette tips. During Cell Lysis when I was adding 10ul of PK to each tube, I completely forgot to switch out the pipette tips.

Our first PCR reaction was set up on 10/16/25 and was a duplex reaction because we both used both Arthropod CO1 and Wolbachia 16S primers together. An annealing temperature of 49 degrees Celsius was used. This Taq polymerase: New England Biolabs One Taq Hot Start Quick-Load 2X Master-Mix with Standard Buffer (#M0488S) was used.

• Our first gel picture was 10/30/25
• It was run at 125 volts for 30 minutes
• Use New England Biolabs 1 kb Plus DNA Ladder for Safe Stains (product #N0559S)

• Second PCR reaction 10-30-25
• Used Taq polymerase as the first PCR reaction
• Single with the Wolbachia primer
• Annealing temp – 55 degrees Celsius
• Made sure to switch out each pipette tip this time

• Our second gel image was taken on 11/6/25
• It was run at 125 volts for 30 minutes
• Use New England Biolabs 1 kb Plus DNA Ladder for Safe Stains (product #N0559S)

Results

I was highly confident that my arthropod would not be Wolbachia infected and I was correct. I know that I can trust these negative Wolbachia results because of my gel images. After the second PCR round I can see that my positive DNA and positive arthropod bands were around 400 bp whereas my arthropod band did not show up. This is direct evidence that my arthropod was negative for Wolbachia.