On September 10th, 2019, at around 1:30 pm, we went to Rock Springs on Mount Tamalpais in Marin County to collect samples of mimulus guttatus. We found populations in creek like areas that still had minimal amounts of water. We collected small samples of this plant and placed them in silica gels to absorb moisture and preserve the plant.
Protocol for DNA extraction from animal tissue
Reagents: We used 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
- We recorded the information about our samples on the “Animal Tissue DNA Extraction” data sheet. Each sample was given a unique ID code and wrote the species name that it was labeled as.
- We put on gloves.
- We labeled one 1.5 ml Screw-cap or Locking Lidmicrocentrifuge tube for each of our samples with the unique ID code.
- We cut out small squares of tissue from each of our samples using a razor blade, scissors, or scalpel onto a paper plate. We made sure to clean the cutting utensil with Ethanol between specimens and cut specimens on different parts of plate.
- We weighed one sample on the scale using a weigh boat to measure ~ 2 – 10 mg of sample tissue. We then estimated the size of each subsequent sample to be the same as the measured sample.
- We added 100 ml of Extraction Solution (ES) to each of our labeled sample tubes (using a p200 µl micropipette and unfiltered tips).
- We added 25 ml of Tissue Preparation Solution (TPS) to the microcentrifuge tubes with the 100 ml of Extraction Solution (ES) and micropipette up and down to mix (use a p200 µl micropipette and unfiltered tips). Pipeting gently.
- We then added each sample to its corresponding extraction microcentrifuge tube using forceps.
- We used a disposable non-filtered pipette tip to gently mash our tissue sample up by hand.
- We incubated the sample at room temperature for 10 minutes.
- We then moved our samples to the heat block to incubate at 95o C for 3 minutes.
- We took the samples out of the heat block and added 100 ml of Neutralizing Solution (NS) (using a p200 pipette and filtered tips). We mixed it by using a vortex.
- Lastly, we put our samples on ice, relabeling if necessary.
Procedure for Amplifying CO1 from Fish
To amplify COI from our fish DNA, we used the components that come in the same kit we used to isolate genomic DNA (gDNA). The Sigma REDExtract-N-Amp Kit includes a solution for use in PCR amplification that includes Taq,dNTPs, and buffers that work optimally given the reagents that are still in our gDNA sample from the isolation procedure.
One factor that improves the chances that PCR will work is the concentration of the template in the reaction. Too much DNA can cause reactions to fail. To prevent this we started by diluting the gDNA we isolated by10-fold (10x).
WE USED FILTER TIPSFOR ALL PCR STEPS
Diluting your gDNA
To make a 10x dilution of our gDNA:
- We labeled a microcentrifuge tube on our bench with “1:10” and the name of our samples.
- We added 18 ml of purified water to the tube we just labeled.
- We then added 2 ml of our gDNA to the tube.
- We gently flicked the tube with our finger to mix the solution.
The PCR reaction
Each of our PCR reactions included the following reagents and volumes:
Water (PCR Quality – autoclaved, filtered) 6.4ml
REDExtract-N-Amp PCR rxn mix 10 ml
Forward Primer FbcF 0.8ml
Reverse Primer FbcR 0.8ml
Tissue Extract (gDNA) (1:10 dilution) 2ml
Total Volume 20ml
We set up a master mix to provide a solution for all of our PCR reactions.
The master mix recipe is:
Reagent Volume (1x) Master (volume x 18)
Water (PCR Quality) 6.4ml 115.2ml
REDExtract-N-Amp PCR rx mix 10 ml 180 ml
Forward Primer 0.8ml 14.4ml
Reverse Primer 0.8ml 14.4ml
Tissue Extract (gDNA- 1:10 dilution) 2 ml ———
Total Volume 20 ml 324 ml
- We wrote the labels of our gDNA sample on our PCR tubes on the top and on the side just below the lid.
- We added 2 ml of the 1:10 dilution of our gDNA to each of our PCR tubes, except the negative control. We were sure to change tips between samples.
- We then pipetted 18 ml of the master mix into each of our PCR tubes, including the negative control.
We left this reaction on ice next to the thermocycler until all PCR reactions were set up. We put the PCR tubes (all samples and the negative control) in the thermocycler and started the reaction, which took between 1.5-2 hours. Our PCR reactions were placed 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
The sushi was purchased on September 2nd, 2019 at 6:17 pm. The sushi was purchased from Kiki Sushi. The samples were stored in the refrigerator until lab.
|Number||Unique ID code||Restaurant Species Name||DNA Barcode Species name|