Bonito Fish Lab Report
INTRODUCTION:
Identification of species and origin based on morphological features by using DNA barcoding technique is an important application for molecular biology and genetics. In this study, we used special fish called bonito fish. These fishes generally live in relatively warm and hot seas and grow until they reach a length of about 30 inches. Bonito fish have a long pectoral fin and two dorsal fins. Also, they have a forked tail.
Animal species and their origins can be detected accurately and quickly with the help of the technique called DNA barcoding (Hanner et. al 2011). Only one gene sequence called COI (mitochondrial c oxidase subunit) gene can be enough to differentiate all animal species. (Hebert et. al, 2003). Differences …show more content…
METHODS:
DNA extraction:
Total DNA was extracted from bonito fish muscle tissue. We used PureLink™ Genomic DNA Mini Kit (ThermoFisherScientific) for DNA extraction. Firstly, we did tissue lysis with lysis buffer to release cellular contents. Then we performed two wash steps to remove salt and unwanted components of cell. Finally, we did elution. After DNA extraction protocol, we used nanodrop-spectrophotometer to determine concentration and purity of our product.
Gel electrophoresis:
We performed agarose gel electrophoresis to separate DNA fragments by size. Since DNA has a negative charge, it migrates towards the positively charged site presence of an electric current. Agarose solution is prepared by agarose powder and TAE buffer. Ethidium bromide (EtBr) which is kind of mutagen is added for track DNA on a gel. EtBr binds DNA and makes it visible under the UV light. Then, the electric current applied at 110 volts for 25 minutes. In the end, we observed our gel in gel imaging machine and under the UV lights.
Polymerase Chain Reaction (PCR) of COI for use in DNA …show more content…
To determine reagents volumes in master mix, we multiplied normal volumes by 11. This number was determined by desired reaction number and plus two reactions. Additional reactions were one for negative control and one for pipetting errors. In the negative control reaction, we used additional sterile ddH2O instead of using DNA template and negative control helped us to confirm contamination of our PCR reagents. The thermal cycles conditions of PCR reactions are as follows: In the first step, we separated or denatured two strands at 94oC for 30 seconds (35 cycles). In the second step, The COI gene which was 605 base pair was amplified with the indicated primers (Wand et.al,
Identification of species and origin based on morphological features by using DNA barcoding technique is an important application for molecular biology and genetics. In this study, we used special fish called bonito fish. These fishes generally live in relatively warm and hot seas and grow until they reach a length of about 30 inches. Bonito fish have a long pectoral fin and two dorsal fins. Also, they have a forked tail.
Animal species and their origins can be detected accurately and quickly with the help of the technique called DNA barcoding (Hanner et. al 2011). Only one gene sequence called COI (mitochondrial c oxidase subunit) gene can be enough to differentiate all animal species. (Hebert et. al, 2003). Differences …show more content…
METHODS:
DNA extraction:
Total DNA was extracted from bonito fish muscle tissue. We used PureLink™ Genomic DNA Mini Kit (ThermoFisherScientific) for DNA extraction. Firstly, we did tissue lysis with lysis buffer to release cellular contents. Then we performed two wash steps to remove salt and unwanted components of cell. Finally, we did elution. After DNA extraction protocol, we used nanodrop-spectrophotometer to determine concentration and purity of our product.
Gel electrophoresis:
We performed agarose gel electrophoresis to separate DNA fragments by size. Since DNA has a negative charge, it migrates towards the positively charged site presence of an electric current. Agarose solution is prepared by agarose powder and TAE buffer. Ethidium bromide (EtBr) which is kind of mutagen is added for track DNA on a gel. EtBr binds DNA and makes it visible under the UV light. Then, the electric current applied at 110 volts for 25 minutes. In the end, we observed our gel in gel imaging machine and under the UV lights.
Polymerase Chain Reaction (PCR) of COI for use in DNA …show more content…
To determine reagents volumes in master mix, we multiplied normal volumes by 11. This number was determined by desired reaction number and plus two reactions. Additional reactions were one for negative control and one for pipetting errors. In the negative control reaction, we used additional sterile ddH2O instead of using DNA template and negative control helped us to confirm contamination of our PCR reagents. The thermal cycles conditions of PCR reactions are as follows: In the first step, we separated or denatured two strands at 94oC for 30 seconds (35 cycles). In the second step, The COI gene which was 605 base pair was amplified with the indicated primers (Wand et.al,