Extraction, but also Polymerase Chain Reaction

In our first technical lab meeting, we: 1) extracted DNA from animal tissue and 2) amplified CO1 from fish.

  1. 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

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