Sample information

When collected, arthropod was hanging off a branch of a red oak tree in my backyard (a more rural area). When first collected, color was more gray. After putting in alcohol to preserve, turn more of a dark red color.

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 30 minutes in a hot water bath at 56 degrees C

Our first PCR reaction was set up on 10/14/25. It was a duplex reaction because we used both the Arthropod CO1 and Wolbachia 16S primers together. The annealing temperature was 49 degrees C. We used this Taq polymerase: New England Biolabs OneTaq Hot Start Quick-Load 2X Master Mix with Standard Buffer (#M0488S). The only difference from written protocol was that we used 88 ul of Taq Master Mix instead of 87.5.

Our first Gel image was taken on 10/21/25 and was run at 125 volts for 22 minutes. The DNA Ladder used was New England Biolabs 1 kb Plus DNA Ladder for safe stains (product #N0559S)

Our second PCR reaction was set up on 10/28/25, used the same taq polymerase as the first PCR reaction and: was a single PCR reaction that used the wolbachia 16s primers, used an annealing temperature of 55 degrees Celsius, the only thing we did differently from the standard protocol is that we used 88 ul of taq polymerase.

Our second gel image was taken on 11-4-25 and was run at 125 volts for 27 minutes. The DNA Ladder used was New England Biolabs 1 kb Plus DNA Ladder for Safe Stains (product #N0559S)

Results

After doing 2 rounds I have a higher confidence level since its showing wolbachia is present for the second time and had a very clear band. The only thing that would change my confidence is the fact that the positive arthropod control didnt show up.