In our first technical lab meeting, we: 1) extracted DNA from animal tissue and 2) amplified CO1 from fish.
- DNA Extraction
-First, I recorded the information about my four fish samples. I assigned the samples ID codes so they could be easily recognized. I labeled them as follows:
CMN01–tuna
CMN02–albacore tuna
CMN03–yellowtail
CMN04–mackerel
-I then put on gloves to reduce the risk of cross contamination and to use proper PPE.
-I labeled one 1.5 mL screw-cap micro centrifuge tube for each sample, and I labeled each tube with a unique ID code (on both the top and side).
-I cut very small portions of fish from each sample with a scalpel, and then weighed them to insure that they were between 2 and 10 milligrams.
-Next, I added 100 micrometers of Extraction Solution and 25 μl of Tissue Preparation Solution to each of the tubes with a p200 μl micropipette and unfiltered tips.
-I also used a 200 μl micropipette and filtered tips to pipette up and down, thereby mixing the contents of each tube.
-I added each fish sample to the tube with its corresponding code.
-I mixed each tube by manually mashing the contents with a non-filtered pipette.
-I incubated the sample at room temperature for 10 minutes.
-I used the heat block to incubate the sample at 95° C for 3 minutes. I set a timer to be as precise as possible.
-I added 100 μl of Neutralizing Solution with a 200 μl micropipette and filtered tips.
-I mixed up the samples by vortexing (for about 10 seconds each).
-I then put my samples on ice.
2. Amplifying CO1
Diluting gDNA
-I labeled four microcentrifuge tubes with “1:10” and their ID codes (on both the top and side).
-I added 18 μl of purified water to each tube.
-I added 2 μl of gDNA from each fish sample to its corresponding tube and I flicked the tubes with my finger to mix the solution.
The PCR Reaction
-We created a master mix for sixteen PCR reactions. This contained the following components:
-I wrote the labels for the gDNA samples on each PCR tubes (on both the top and side).
-I added 2 μl of the 1:10 dilution of the gDNA to each PCR tube, except the negative control.
-I pipetted 18 μl of the master mix into each PCR tube, including the negative control.
-Finally, I placed the PCR tubes on ice.
-The PCR tubes were placed in the thermocycler for 1.5-2 hours.
-Finally, all the PCR tubes were placed in the freezer.
Settings for the thermocycler:
94° C – 4 min (initial denaturation)
30 cycles of:
94° C for 30 sec (denaturing)
52° C for 40 sec (annealing)
72° C for 1 min (extension)
72° C for 10 min (final extension)
10° C hold