Methods

overview: collecting molecular data

We collected molecular data using Sanger sequencing. This provided us with the nucleotide base order in the individuals targeted DNA sequence.

STEP 1: Extraction

DNA was extracted from the femur of each individual specimen using enzymatic digestion with proteinase K.

Step 2: Polymerase Chain Reaction (PCR)

Process of Polymerase Chain Reaction performed.

Polymerase Chain Reaction (PCR) was used to amplify the COII gene region of the mitochondrial DNA

Step 3: Gel Electrophoresis

Process of Gel Electrophoresis performed.

Gel electrophoresis was performed to confirm correct amplification and duplication of the targeted gene. Each sample was stained and run alongside a DNA ladder. The negatively charged DNA fragments migrate down the gel to the positive side

Step 4: Automated Sequencer

The amplified DNA was then sequenced using an automated sequencer to determine the nucleotide base pairs and their order

Step 5: Visualizing the Molecular Data

DNA sequences were use to create a phylogenetic tree using the R package XXX.

Closely related species (Arphia simplex, Meteor nevadensis, Anconia integra and Chortophaga australior) were used as outgroups.

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