Authornpdibanez

Lab 6 – Intro to Geneious

This week, we worked with the program Geneious to sequence our fish DNA.  Unfortunately, the fish samples that I PCR’d did not produce a useable barcode sequence.  For this lab, I used barcodes from a previous student.  The samples were EZ02 and EZ03.  When she collected the sushi, the student was told that EZ02 was red snapper and EZ03 was yellowtail.  I used Geneious to assemble, edit and BLAST these DNA sequences, and found the results of the species barcodes.  EZ02 was not red snapper as advertised, it was Oreochromis niloticus, or Nile tilapia.  The other sample, EZ03 was Seriola quinqueradiata, or yellowtail, so this one was correctly labeled.

 

The EZ02 alignment I built had only one polymorphism, in column 663.  The EZ03 alignment had seven polymorphisms, found in columns 5, 6, 7, 11, 12, 15, and 201.

Lab 5: Mimulus Encounters Part 2

This week, the molecular ecology lab took a field trip to Marin County to visit more wild populations of our focal plant species, Mimulus guttatus(=Erythranthe guttata).

 

On Tuesday, September 24, 2019 at 1:00PM, the class left in two passenger vans, and headed to Red Rock Spring, Marin County.  The site is located at a gravel pull-out on the east side of Highway 1, just South of Stinson Beach.

 

After getting lost on a Google-maps detour, we found the gravel parking lot and joined the rest of the class. We observed a natural spring, flowing out of PVC pipes from the hillside, and the population of Mimulus guttatus growing in the spring-fed puddle.  The population had many more flowers on display than we had seen at the populations from the Lab 3 field trip, two weeks prior.  All of the flowers appeared to have closed stigmas, suggesting that they had been pollinated.  The habitat was a steep, disturbed hillside that had been carved away to make room for Highway 1.  The population, although fed, seemingly perennially, by the stream, was in full sun, and we can vouch for the heat stress.

 

After leaving this population, the vans left and headed south along Highway 1, eventually turning east and arriving at the Redwood Creek Trailhead.  The trail crosses Redwood Creek, a perennial creek that flows through Muir Woods, all the way to Muir Beach.  The class followed the trail until we got to an old wooden bridge that crosses the creek.  We veered to the right, down the horse bypass trail.  Once we got to the creek, we followed it upstream, searching for Mimulus guttatus.  The creek is surrounded by a dense riparian corridor, a continuous canopy, and characterized by a gravelly, sandy substrate in the creek bed.

 

By now, the class recognized the familiar habitat, and expected to see our monkey flower growing near the waters’ edge.  After ducking under vegetation, crossing log bridges, and only a few submerged shoes, I spotted a population of Mimulus on a dry gravel bar along the edge of the creek.  The individuals were numerous, small, vegetative rosettes.  About 100 ft further upstream, the class found another populations of small Mimulus rosettes.  These populations had not yet flowered.  We considered the likelihood of these plants flowering before winter storms drown them out or wash them away.  We also pondered the movements of pollinators, and how that might affect gene flow between the populations we’ve seen in the field.

 

Eventually we made our way back along the creek, and headed back to campus.

 

Note: Sorry about the absence of photos, my phone was dead before arriving at the first population.

Lab Entry 4

Week 4 lab was an extension of the Sushi Lab started in Week 2.

 

Gel Electrophoresis

  1. We removed our PCR tubes with our samples of fish tissue from ice and given time to thaw.
  2. We laid out 15 dots of about 1 microliter of loading dye on a sheet of parafilm.
  3. We recorded the names and order of samples on a piece of paper.
  4. Each lab member pipetted 3 microliters of each PCR product onto a single dot of loading dye, changing pipette tips in between each sample.
  5. We loaded each dot of loading dye mixed with PCR product into a cell in the gel setup, including a control in the first cell and a ladder in the 15th
  6. We fastened the lid to the gel and turned on the machine to run at 130 volts for 30 minutes.

 

Clean-Up of PCR products for sequencing – ExoSAP

  1. We determined the number of PCR clean-ups for our table, to prepare sufficient master mix. This included one for each sample, and a few extra, for a total of 18.
  2. The master mix recipe was prepared as follows:

Master Mix:                                       Per Rxn              Total Rxn: 18

H2O                                                    10.59 uL    _____190.6_______

10x buffer (Sap 10x)                      1.25 uL      _____22.5______

SAP                                                    0.44 uL      ______7.9________

Exo                                                     0.22 uL      ______4.0______

 

Master Mix Total                     12.5 uL               _______225_______

 

PCR   Product                                       7.5 ul

Total Cleaned-up Volume        20.0 uL

 

3.  We put each reagent in the ice bucket on the table.

4.  We pipetted 7.5 uL of each PCR product into a clean, labeled 0.2 uL PCR tube.

5.  We made the ExoSap master mix, keeping the reagents on ice while it was made.

6.  We pipetted 12.5 uL into each PCR product tube.

7.  We placed the tubes in a thermocycler and started the EXOSAP program.

8.  After the program was done (~ 45 minutes later), Dr. Paul placed the PCR tubes in a labeled tube rack and placed them in the freezer.

 

Lab 3 – Mt. Tamalpais Fieldtrip

This week, the molecular ecology lab took a field trip to Mt. Tamalpais to visit several wild populations of our focal plant species, Mimulus guttatus (=Erythranthe guttata).

 

On Tuesday, September 10, 2019 at 1:00PM, the class left in two passenger vans, and headed to the Rock Springs Trailhead on Mt. Tamalpais, Marin County.  To get to the site, we left San Francisco on 101 North, crossing the Golden Gate Bridge into Marin County.  We took exit 450B and merged onto Sir Francis Drake Blvd.  We turned left onto Pacheco Avenue, then right onto Broadway, and then left onto Bolinas Rd.  We followed Bolinas Rd for approximately 10 miles, and then took a left on West Ridgecrest Blvd.  Four miles up this road, the parking lot is on the right.

 

After parking in the lot, the class headed northwest on the Cataract Trail.  This trail soon led us to a bridge over Cataract Creek.  The class paused here, under a dense oak riparian canopy, to observe a population of Mimulus guttatus growing in the dry creek bed.  The plants were in their vegetative state, forming mats of rhizomatous rosettes, which were patchily distributed along the creak bed.  We talked about their morphological variation, and the research that Dr. Paul’s grad student, Alec Chiono, is conducting about the climate variability hypothesis using M. guttatus.

Figure 1.  Dry bed of Cataract Creek with M. guttatus plants growing in it.

 

The class spread out here to collect samples of M. guttatus leaves.  We each tried to collect distinct individuals, based on spatial separation.  We each took a small sample of our plant, approximately 2-3 leaves of new growth, and dropped them into tubes full of silica powder.  We immediately inverted the tubes several times to make sure the sample was covered in silica, to help dry out the tissue.  We labelled the tubes with our initials, the date, and the population code CATB.

Figure 2. Collection vial with M. guttatus sample and silica in it.

 

After each person made their collection, we walked back down the trail toward the parking lot, but diverted north at the trail split to observe a population of M. guttatus along a different stretch of Cataract Creek.  This population’s habitat lacked the dense canopy, and instead occurred in a sunny, nonnative grassland.  The Mimulus plants in this location were mostly brown and drying out, but some fruits were left on the stalk, full of seeds for us to observe.  Dr. Paul crushed some of the fruits, exposing tiny black seeds.  We discussed dispersal hypotheses for this species.

 

Next we went back to the cars and drove back down West Ridgecrest Blvd for a few minutes, before pulling off on the side of the road, just past a gate on the left.  We headed to the west side of the road, to observe a serpentine outcrop.  We discussed the harsh, dry characteristics of this substrate and the general ecology of species that occur here.  Not many species occurred in this rocky area (mostly Arctostaphylos sp. and Adenostoma fasciculatum around the edges).  I observed a few dried up individuals of the genus Streptanthus.

Figure 3. Serpentine outcrop on a hillside off W Ridgecrest Blvd.

 

We crossed the street, and walked past a gate to an old mining area.  On an exposed serpentine outcrop near the trail, we observed another population of M. guttatus. Here, it could be seen that the M. guttatus plants grew along a small drainage, where water flows ephemerally along the surface of the rock.

Figure 4.  M. guttatus population on a serpentine outcrop.

 

We next headed back down West Ridgecrest Blvd to another trailhead.  Walking west on this trail for about 5 minutes, we got to an intermittent creek with a small riparian corridor, running down an otherwise dry, grassy hillside.  The riparian corridor, with its dense green vegetation was contrasted sharply by the surrounding dry grass.  In this drainage, running with shallow water, a population of M. guttatus was in bloom.  We observed the flowers and discussed potential pollinator hypotheses.

Figure 5.  M. guttatus in flower in a wet creek bed.

 

This completed our tour of the variable habitats of M. guttatus.  After enjoying the view and the fresh air for a bit longer, we headed back to campus.

Figure 6.  A view of the city from up high on Mt. Tamalpais.

Lab 2: The Sushi Test

Sushi Collection:

I collected my raw fish samples from Kufuya Japanese Restaurant at 7001 Geary Blvd, San Francisco, CA at 7:30 PM on Monday 2 September 2019.  I ordered a plate of nigiri (raw fish on top of rice) with three different species of fish: yellow tail, white tuna, and red snapper.  I cut a small piece of each species of fish and stored each piece in a 2.0 ml tube provided in class.  I labeled each tube with a unique code and recorded which code corresponds to each species of fish.  I stored the tubes with fish samples in my home refrigerator over night, and then transferred them to an ice bucket in lab the following day.

Figure 1. Photograph of assorted nigiri plate as served. Fish species ordered (from left to right): yellow tail, white tuna, red snapper

DNA Extraction:

Reagents: I 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
-1.5 ml microcentrifuge tubes
-Razor blades/scissors/scalpels
-Heat block
-Vortex
-Ice
-Sharpie
-Gloves
1. I recorded the ID code and species common name for each of my samples on the “Animal Tissue DNA Extraction” data sheet. I gave each sample a unique ID code, starting with my initials (NI) and then a number (01-03). I recorded the species name that my samples were labeled as on the menu.  The codes correspond as follows:
Unique ID Code                    Restaurant Species Name
 NI 01                                         Yellow Tail
 NI 02                                        White Tuna
 NI 03                                        Red Snapper
2. I wore gloves and other appropriate PPE for the remainder of the DNA extraction procedures.
3. I used a sharpie to label one 1.5 ml Locking Lid microcentrifuge tube for each of my samples with the unique ID code. I wrote the unique ID code BOTH on the side and on the top of the tube.
4. I took each sample out of its tube, and placed on a labeled paper plate to cut out a small piece. Using a scalpel, I carefully cut out a small square of fish tissue. I made sure to cut away any portion that was in contact with the rice and used tissue from inside the sample. In between samples, I cleaned the scalpel with Ethanol, and cut specimens on different parts of plate, which were labeled.
5. I put a weigh boat on a scientific scale, and zeroed the scale.  Then I added a sample of yellowtail to weigh the sample.  I cut the sample smaller, and reweighed until I had a piece that was approximately 10 mg.  I then cut similar size pieces of the other samples, using the yellow tail sample to estimate the weight of the subsequent samples.
6. I added each 10 mg sample to its corresponding extraction microcentrifuge tube using forceps, cleaning the forceps in-between each sample.
7. I added 100 µl of Extraction Solution (ES) to each of my labeled sample tubes using a p200 μl
micropipette and unfiltered tips.
8. I added 25 µl of Tissue Preparation Solution (TPS) to each of  the microcentrifuge tubes with the 100 µl of Extraction Solution (ES) and micropipetted up and down to mix using a p200 μl micropipette
and unfiltered tips.
9. I used disposable non-filtered pipette tips to gently mash my tissue samples by hand.
10. I incubated the samples at room temperature for 10 minutes.
11. I then moved my samples to the heat block to incubate at 95oC for exactly 3 minutes.
12. After 3 minutes, I removed the samples from the heat block. I then added 100 μl of Neutralizing Solution (NS) to each tube using a p200 pipette and filtered tips. I mixed each sample by vortexing on a vortex machine for ten seconds.
13. I then double-checked that the labels had not been rubbed off the tubes, and put the samples in an ice bucket.

 

Amplifying CO1 for PCR:

Diluting the gDNA
To make a 10x dilution of the gDNA:
1. I labelled a microcentrifuge tube with “1:10” and the unique ID code for each sample from the previous steps. I wrote this on the top and on the side of each of the 3 tubes.
2. I add 18 µl of purified, filtered, sterile water to each of the labeled dilution tubes.
3. I added 2 µl of the gDNA (from the previous steps) of each species to the dilution tubes.
4. I gently flicked the tubes with my finger to mix the solutions.
The PCR reaction
Each of my PCR reactions included the following reagents and volumes:
Reagent                                                                                  Volume
Water (PCR Quality – autoclaved, filtered)               6.4 µl
REDExtract-N-Amp PCR rxn mix                                  10.0 µl
Forward Primer FbcF                                                            0.8 µl
Reverse Primer FbcR                                                             0.8 µl
Tissue Extract (gDNA) (1:10 dilution)                         2.0 µl       
Total Volume                                                                            20 µl
To set up multiple PCR reactions under the same conditions, including my 3 samples as well as the samples for my lab-mates, we made a master mix that included the combined volumes of all of the reagents for multiple reactions, except for the diluted tissue extract. This strategy improves success by minimizing errors due to pipetting small volumes multiple times.
To make enough master mix for the whole table, we used the volumes for the individual reactions (listed
above) multiplied by the number of reactions, plus some extra just in case.  Our table had 13 samples, one negative control, and we wanted to make sure we had a little extra. So we would multiplied the volumes times 13 + 1+ 2 = 16.
A negative control is all the reagents required for a PCR reaction, but NO gDNA template. Thus, if anything is amplified in the negative control, we know there is a source of contaminating gDNA, other than the samples we are trying to PRC amplify.
The additional 2x reagents are needed to account for minor errors in pipetting that could result in
not having enough total volume in the master mix for all individual reactions.
The master mix recipe is:
Reagent                                                                   Volume (1x)      Master (16x)
Water (PCR Quality – autoclaved, filtered)     6.4 µl                  102.4 µl
REDExtract-N-Amp PCR rxn mix                      10.0 µl                  160 µl
Forward Primer FbcF                                                 0.8 µl                   12.8 µl
Reverse Primer FbcR                                                 0.8 µl                   12.8 µl
Tissue Extract (gDNA) (1:10 dilution)               2.0 µl                    —-           
Total Volume                                                               20 µl                  290 µl
Finishing up:
1. I wrote the labels of my gDNA sample on PCR tubes in sharpie (on the top and on the side just below the lid).
2. The master volume of each reagent above was added to a tube labelled “MM” for master mix, except for the REDExtract-N-Amp PCR rxn mix.
3. I added 2 µl of the 1:10 dilution of each gDNA dilution to each of the labeled PCR tubes, except the negative control, changing pipette tips in-between each sample.
4. I added the REDExtract-N-Amp PCR rxn mix to the master mix.
5. I then Pipetted 18 µl of the master mix into each of my PCR tubes, including the negative control, changing pipette tips in-between each sample.
6. We set these PCR reaction tubes on ice next to the thermocycler until all the PCR reactions were set up. We then put the PCR tubes (all samples and the negative control) in the thermocycler and started the reaction, which takes between 1.5-2 hours.
7. When the PCR reactions were finished with the therm-cycling (see settings below), they were placed in the freezer.
Settings for the thermocycler:
94oC – 4 min (initial denaturation)
30 cycles of:
      94oC for 30 sec (denaturing)
      52oC for 40 sec (annealing)
      72oC for 1 min (extension)
72oC for 10 min (final extension)
10oC hold

Figure 2. Photograph of my lab table’s DNA extraction gel electrophoresis results. My three samples are labeled NI01, NI02, and NI03.

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