0

Lab 6 Entry

Using Geneious, we were able to match our fish DNA to DNA in the database. To do this we first copied the reverse sequence from the ‘Fish barcode Reverse Reads’ folder and pasted it into the ‘Forward Reads’ folder. Then, we selected the forward and reverse sequences of the same ID and clicked the Align/Assemble tab, choosing ‘De novo assembly”. Using the default settings, a new file appeared containing the consensus sequence for that fish DNA. We edited the sequence to clean up any discrepancies and saved these changes. We then right-clicked this file and chose ‘Generate consensus sequence’. We used this file to BLAST our sequence by right-clicking the file and selecting ‘BLAST’, keeping the default settings. Another file appeared, allowing us to see a set of top matches for our sequence. These matches told us what species our fish DNA most closely resembles. We created a new folder for a fish barcode test alignment and selected the assembly consensus sequence and 5-10 hits from the BLAST search, pasting these into the new folder. We then selected all of these sequences in the new folder and right-clicked, choosing ‘Multiple Align’ and then ‘Muscle Alignment’ with default settings. A new file was generated, showing polymorphisms between our DNA and other DNA of the same species.

Unfortunately, only two of my DNA barcodes were successful: KRS1 and KRS4.

Sushi Samples
Number Unique ID code Restaurant Species Name DNA Barcode Species Name
1 KRS1 Tuna Thunnus Albacares (Yellowfin Tuna)
2 KRS2 Mackerel N/A
3 KRS3 Albacore N/A
4 KRS4 Salmon Salmon Salar (Atlantic Salmon)

The successful DNA barcodes matched with the species specified at the restaurant.

For KRS1, there were 11 polymorphic sites found in columns 31, 167, 228, 324, 332, 333, 344, 346, 371, and 387.

For KRS4, there were 10 polymorphic sites found in columns 69, 586, 590, 611, 612, 614, 625, 628, 631, and 632.

0

Lab 5 Entry- Second Field Trip

On Tuesday, September 24, 2019, we traveled back to Mount Tamalpais, this time stopping near Muir Beach to collect and view samples of mimulus guttatus. We stopped at a natural spring to drink the water and observe the flowering mimulus guttatus. We then traveled to a shaded creek bed to find more mimulus guttatus samples. Alec collected these samples to include in our lab.

Mimulus guttatus near a natural mountain spring (pictured behind the flower).

Mimulus guttatus flowers.

Mimulus guttatus in a creek bed.

0

Lab 4- Gel Electrophoresis/ PCR Clean-up

To begin the electrophoresis of PCR product:

  • First, we thawed our PCR tubes
  • We dotted 16 loading dye dots (~1 ul) on a sheet of parafilm
  • We pipetted 3 ul of each PCR product on its own dot
  • We then loaded all dots into the gel (setting the pipette to ~5ul)
  • We ran the gel at 130 volts for 30 min

My PCR products were placed in lanes 9-13, beginning with KRS1 and ending with the negative control, respectively.

Clean-up of PCR products for sequencing- ExoSAP

  • First, we labeled new 0.2 ul PCR tubes with each of the sample codes
  • We then made the ExoSAP Master Mix
  • Lastly, we placed the PCR tubes into a thermocycler

ExoSAP PCR Clean-Up Protocol 

Recipe to clean-up one PCR reaction of 7.5 uL

Master Mix:                                  Rxn: 1          Rxns: 18

H2O                                                10.59 uL            190.62 uL

10x buffer (Sap 10x)                      1.25 uL             22.5 uL

SAP                                                0.44 uL               7.92 uL

Exo                                                0.22 uL               3.96 uL

Master Mix Total                    12.5 uL              225 uL

PCR Product:

PCR                                                7.5 ul

Total Cleaned-up Volume   20.0 uL

Steps:

  • We determined the number of PCR clean-ups
  • We calculated the volume of Master mix needed
  • We put reagents on ice
  • We pipetted 7.5 uL of each PCR product into a clean, labeled 0.2 uL PCR tube
  • Then we made the ExoSap master mix, keeping the reagents on ice while it was made
  • We pipetted 12.5 uL into each PCR product tube
  • We placed the tubes in a thermocycler and start the EXOSAP program
  • After the program completed (~ 45 minutes), we placed the PCR tubes in a labeled tube rack and placed them in the freezer

    DNA barcode for our group, Queen Salmon.

PCR results after 16 hrs 5 min.

0

Population Genetics Project

Mimulus guttatus in a creek bed.

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.

0

Sushi Lab Protocol

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)

 

Materials:

  • p200 microcentrifuge
  • 5 ml microcentrifuge tubes
  • Razor blades/scissors/scalpels
  • Heat block
  • Vortex
  • Ice
  • Sharpie
  • Gloves

 

  1. 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.

 

  1. We put on gloves.

 

  1. We labeled one 1.5 ml Screw-cap or Locking Lidmicrocentrifuge tube for each of our samples with the unique ID code.

 

  1. 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.

 

  1. 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.

 

  1. We added 100 ml of Extraction Solution (ES) to each of our labeled sample tubes (using a p200 µl micropipette and unfiltered tips).

 

  1. 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.

 

  1. We then added each sample to its corresponding extraction microcentrifuge tube using forceps.

 

  1. We used a disposable non-filtered pipette tip to gently mash our tissue sample up by hand.

 

  1. We incubated the sample at room temperature for 10 minutes.

 

  1. We then moved our samples to the heat block to incubate at 95o C for 3 minutes.

 

  1. 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.

 

  1. 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:

  1. We labeled a microcentrifuge tube on our bench with “1:10” and the name of our samples.

 

  1. We added 18 ml of purified water to the tube we just labeled.

 

  1. We then added 2 ml of our gDNA to the tube.

 

  1. 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:

Reagent                                                            Volume

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

 

Finishing up:

  1. We wrote the labels of our gDNA sample on our PCR tubes on the top and on the side just below the lid.

 

  1. 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.

 

  1. 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

0

Sushi Lab

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.

Sushi Samples
Number Unique ID code Restaurant Species Name DNA Barcode Species name
1 KRS 1 Tuna
2 KRS 2 Mackerel
3 KRS 3 Albacore
4 KRS 4 Salmon

From left to right: Tuna, Salmon, Albacore, and Mackerel

Location and time of purchase.