DNA Barcoding

Introduction

On the 6th September, we extracted the DNA from our raw fish that we collected. The purpose was to find out if the raw fish we got was actually what is was advertised to be by analyzing the fish DNA.

Methods

We cut up our raw fishes into tiny pieces, weighing approximately 2mg and labelled screwcap microfuge tubes. We used a commercial DNA extraction kit for this extraction. We pipetted 100 microlitres of extraction solution into each tube and then added another 25 microlitres of tissue preparation solution. We then added the fishes into the microfuge tubes that has their label (i.e. salmon in the tube labelled as salmon). We used a disposable pipette tip to smash the fish in the tube and left it out for around 10 minutes at room temperature. After that, we moved it to a heat block for 3 minutes (95 degrees celcius). Once we removed it, we added 100 microlitres of neutralizing solution and used a vortex machine to mix it up. We then left it in ice while we prepared our master mix (a mixture of water, PCR rxmix, forward and reverse primer). We diluted our fish samples by pipetting 18 microlitres of purified water in a microcentrifuge tube and then another 2 microlters of our fish DNA. In a PCR tube, we added 2 microlitres of our DILUTED fish DNA and 18 microlitres of our master mix and started out PCR.

The next week, we ran two gel electrophoresis on our fish DNA, one for our gDNA and one for our PCR product. The agarose block was prepared for us already (well for the first one) and all we had to do was pipette our sample into the wells and run the gel electrophoresis for about 10 minutes at 145V. To prepare out samples, we pipetted 2 microlitres of loading dye on a piece of parafilm and pipetted 3 microlitres of our DNA onto it. We repeated this step for our PCR product BUT we reused our old agarose gel by melting it and letting it cool and set. After determining which PCR worked, we did an Exosap PCR Clean up. We first made a master mix per table consisting of water, buffer, Exo and SAP, and then we pipetted our PCR product into a PCR tube and pipetted 12.5 microlitres of our master mix in the tube. After this, we put the tubes in a thermocycler and ran the program and after it’s done, we put it in the freezer.

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