Molecular Ecology – DNA barcoding and its use for species identification and food traceability

Molecular Ecology – DNA barcoding and its use for species identification and food traceability

 

Some of the chemicals used in the DNA extraction process are toxic and therefore should be handled with caution. A lab coat and gloves should be worn at all times. Washing your before and after you have finished the practical is highly recommended.

Materials

                     2x 1.5ml microfuge tubes

                     1x DNA spin column within a collection tube

                     1x Tips Bin

                     P1000 pipette and 1 box of 1000μl tips

                     P200 pipette and 1 box of 200μl tips

                     P10 pipette and 1 box of 10μl tips

                     Water bath (set to 56°C)

                     Bench-top microfuge

                     Vortex Mixer

                     Gloves

                     Indelible fine marker (black)

                     Buffer ATL*

                     Proteinase K*

                     Buffer AL*

                     Ethanol*

                     Buffer AW1*

                     Buffer AW2*

                     Elution Buffer AE*

 

*these reagents are shared between groups of 6 students at the half of the bench, be careful when pipetting them to use the exact amount detailed in the protocol.

 

Bench locations are numbered 1-44, THIS WILL BE THE NUMBER THAT IDENTIFIES YOUR SAMPLE, i.e. the one you need to label your tubes with. Write down the species and your sample number in your notebook, we will need it to check the correct species identification.

There are small chunks of fish from a commercial package of fish pie. Pick up one per person. When you collect your sample for DNA extraction take into account that less is more; it is important to get a small amount of the sample. When working with fish (or other animals) the recommended amount of sample is around 10 to 20 mg, which represents a very small amount of sample about this size: 

DNA Extraction and PCR Preparation (tick each one of the steps after you have done it)

1.      Use a scalpel to remove a small tissue sample (~3mm) from the fish muscle. 

2.      Place the tissue in a fresh 1.5μl microfuge tube labelled with your sample number.

3.      Add 180 μl of buffer ATL (lysis buffer) so it covers the sample completely

4.      Add 20 μl of Proteinase K and vortex briefly.

5.      Incubate the sample in the buffer in the water bath for approx. 30 mins at 56°C. 

6.      After the 30 min incubation, vortex 15s before proceeding to step 7

a.      What does the tissue look like now? Why?

1.      After the 10 min take your tube and add 200 μl of ethanol (labelled EtOH). Mix by vortexing. (*) If at this stage there is a chunk of tissue in your tube, ask a demonstrator, briefly spin it and take only the supernatant for the next stage.

a.      What does the sample looks like now?

2.      Pipette the mixture (or supernatant) into the spin column placed in a 2ml collection tube. Remember to label it with your number. (Ensure you don’t touch the white filter with the tip as this would damage the column and you would lose the DNA)

3.      Centrifuge at 8000 rpm for 1 minute.  This will force the liquid through the silica column whilst retaining the DNA. Discard the liquid from the collection tube and reuse the tube.

4.      Add 500μl of wash buffer AW1 and centrifuge at 8000 rpm for 1 min. Discard the liquid in the collection tube and reuse the tube.

5.      Add 500μl of wash buffer AW2 and centrifuge at 20,000 rpm for 3 min.  Discard the liquid in the collection tube and reuse the tube.

a.       What are these washes for?

 

 

6.      Discard the collection tube but keep the column and transfer to a clean 1.5 eppendorf tube, labelled with your sample number.

a.       Why are you keeping the column?

7.      Add 100μl elution buffer AE to the center of the column.

a.       What is the Elution buffer for?

1.      Allow elution buffer to stand in the column for 1 minute at room temperature – this step allows the buffer to interact with the DNA, weakening its binding to the column so that when you spin, the DNA falls through into the collection tube.

2.      Centrifuge for 1 minute at 8000 rpm and discard the spin column.

a.       Why do you keep the tube this time?

 

Molecular Ecology – DNA barcoding and its use for species identification and food traceability

 

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