Tuesday, August 27
Last Tuesday, Mikayla and I went to Ginza Sushi on Haight Street and collected samples for lab the following week. We ordered two rolls and sashimi to ensure that we got four different kinds of fish. The first roll, “Bara” contained maguro as the main fish, the other roll “Ginza” had yellowtail and the sashimi bowl included salmon, akami and yellowtail. We ended up not sampling “Ginza” since we already had yellowtail from the sashimi bowl.
To collect the samples we used chopsticks to pick off a piece about the size of an eraser and put it into the tubes that were provided. To avoid contamination (as much as possible), we used new chopsticks and plates each time.
DNA EXTRACTION FROM ANIMAL TISSUE
Reagents: We will be using a commercial DNA extraction kit (Sigma REDExtract-N-Amp Tissue PCR Kit)
- Extraction Solution (labeled ES)
- Tissue Preparation Solution (labeled TPS)
- Neutralizing Solution (labeled NS)
- p200 microcentrifuge
- 5 ml microcentrifuge tubes
- Razor blades/scissors/scalpels
- Heat block
[ Reagents and Materials copied from lab protocol document (JP) ]
- The first step was to assign unique codes to each fish sample. These codes and the fish they were assigned to were noted on the “Animal Tissue DNA Extraction” data sheet provided.
- OY-01 – Salmon
- OY-02 – Akami
- OY-03 – Yellowtail
- OY-04 – Maguro
- Next, we got paper plates, drew lines on it to divide it into 4 spaces to keep the fish samples separate and I cut off a little piece of each- making sure to sterilize the scalpel each time I moved from one fish to another with Kim wipes and ethanol. Each square on the plate was labeled with the code and the fish name and gloves were used as well to avoid contamination of the samples.
- After the little pieces were cut I weighed each one on the scale to ensure it was around 2mg [they all came out to be a little over 2 mg]. Separate weigh boats were used for each sample and the scale was zero-ed each time a new weigh boat was on it.
- Once all the samples were weighed and ready I labeled four screw cap microcentrifuge tubes with the codes from step 1- I labeled the side and the top.
- 100 microliters of Extraction Solution (ES) were then pipetted into each tube using a p200 microliter micropipet. The procedure stated to use unfiltered tips but we could not find any that fit the micropipet so we ended up using filtered tips.
- Next 25 microliters of Tissue Preparation Solution (TPS) was added to the same tubes using the same p200 microliter pipet (and new tip). To mix, I gently pipetted up and down using that same tip and pipet.
- Finally, each sample was put into its designated tube using forceps- the forceps were cleaned before touching each different sample using ethanol.
- Next, using a non filtered tip, I gently mashed each sample in the tubes. Different tips were used for each sample.
- The samples were incubated at room temperature for 10 minutes.
- After incubation, I moved the samples to the heat block. They were incubated at 95o C for 3 minutes.
- After that, I added 100 microliters of Neutralizing Solution (NS) using the same p200 pipette and filtered tips. They were then mixed using the vortex machine.
- Lastly, they were put on ice and the extra fish that was collected was given to Professor in case the experiment needed to be repeated.
AMPLIFYING CO1 FROM FISH
** Everything in this part is using filtered tips **
A. Dilute gDNA
- I labeled 4 microcentrifuge tubes with the unique code names and “1:10” on the top and sides.
- 18 microliters of purified water was added to each tube.
- 2 microliters of each fish sample tube [gDNA] was added to each microcentrifuge tube.
- Then I gently flicked each tube to mix it.
B. PCR reaction
- For the PCR reaction we needed to make a master mix with our lab benches. We had 16 samples total (4 each) so we made a master volume to account for 18 samples so we would have a little extra just in case. The amounts of each reagent are shown below.
- Reagent Volume
- Water (PCR Quality – autoclaved, filtered) 6.4 ml
- Master volume (volume x 18) = 115.2 microliters
- REDExtract-N-Amp PCR rxn mix 10 mlMaster volume (volume x 18) = 180 microliters
Forward Primer FbcF 0.8 ml
Master volume (volume x 18) = 14.4 microliters
Reverse Primer FbcR 0.8 ml
Master volume (volume x 18) = 14.4 microliters
Tissue Extract (gDNA) (1:10 dilution) 2 ml
Total Volume 20ml
Master volume (volume x 18) = 324 microliters
2. Next, we got PCR tubes and labeled each with the codes from earlier. Everyone got 4 tubes except for one of us who got 5 so we used that extra one as a negative control.
3. 2 microliters of the 1:10 dilution of the gDNA was affix to each of the small PCR tubes except for the negative control. Tips were changed between each sample.
4. Next 18 microliters of the master mix were added to each tube, including the negative control. Again, tips were changed between each sample.
5. This was all put on ice until all the PCR reactions in the lab were ready to go. Then we put them all in the thermocycler for the reaction.
6. Professor Paul ran the reaction – settings are shown below and it took about 1.5 – 2hours for the full reaction. They were then put in the freezer when the cycling was complete.
Settings for the thermocycler:
94o C – 4 min (initial denaturation)
30 cycles of:
94o C for 30 sec (denaturing)
52o C for 40 sec (annealing)
72o C for 1 min (extension)
72o C for 10 min (final extension)
10o C hold