# Protocol for mitochondrial DNA isolation from Macrobrachium rosenbergii and Penaeus monodon using differential centrifugation

## TL;DR

This paper provides a detailed protocol for isolating mitochondrial DNA from two crustacean species using differential centrifugation, without commercial kits.

## Contribution

A novel, cost-effective protocol for mitochondrial DNA isolation from Macrobrachium rosenbergii and Penaeus monodon using differential centrifugation.

## Key findings

- The protocol successfully isolates high-quality mitochondrial DNA suitable for sequencing.
- Differential centrifugation effectively enriches mitochondrial fractions without commercial reagents.
- PCR amplification and gel electrophoresis confirm the presence and purity of mt-DNA.

## Abstract

The mitochondrial genome (mitogenome) is a vital tool in molecular phylogenetics, evolutionary biology, and population genetics. Here, we present a protocol for extracting mitochondrial DNA from shrimp (Macrobrachium rosenbergii) and prawn (Penaeus monodon) species using a differential centrifugation approach without relying on commercially available kits. We describe steps for sample preparation, crude mitochondrial extraction, mitochondrial DNA isolation, and purity assessment. We then detail procedures for confirming the presence of the mitogenome and sequencing the mitochondrial genome.

For complete details on the use and execution of this protocol, please refer to Pal et al.1,2

•Instructions for enriching mitochondrial fractions using differential centrifugation•Steps for extracting high-quality mitochondrial DNA•Guidance on confirming mt-DNA using PCR amplification and gel electrophoresis•Procedures for annotating mitochondrial genomes

Instructions for enriching mitochondrial fractions using differential centrifugation

Steps for extracting high-quality mitochondrial DNA

Guidance on confirming mt-DNA using PCR amplification and gel electrophoresis

Procedures for annotating mitochondrial genomes

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The mitochondrial genome (mitogenome) is a vital tool in molecular phylogenetics, evolutionary biology, and population genetics. Here, we present a protocol for extracting mitochondrial DNA from shrimp (Macrobrachium rosenbergii) and prawn (Penaeus monodon) species using a differential centrifugation approach without relying on commercially available kits. We describe steps for sample preparation, crude mitochondrial extraction, mitochondrial DNA isolation, and purity assessment. We then detail procedures for confirming the presence of the mitogenome and sequencing the mitochondrial genome.