Lab 06: An Introduction to Geneious

Allyson Luber

October 1, 2019

Computer Lab: An Introduction to Geneioius

  • In this computer lab we were able to assemble, edit, and BLAST each of our sequence/reverse pairs
  • Allowed us to match the fish species we were supposed to be served at the restaurants
  • Attached below are snapshots of the nucleotide alignments for each species, aligned next to closely related (or the exact species) fish:

AEL02

AEL04

  • Specimen 02 (AEL02) was labeled as “Eel” on the menu and was found out to be matched with Japanese/European eel (Anguilla rostrata voucher; 99.1% grade)
  • Specimen 04 was labeled as “White tuna” and was found to be matched with multiple different tuna families (Thunnus alalunga, Thunnus orientalis)
  • In each alignment there were at least 10 polymorphic sites found (listed below)

02: 25, 76, 81, 105, 110, 123, 172, 196, 211, 241

04: 73, 124, 130, 133, 163, 250, 295, 340 (only 8)

  • Specimens 01 and 03 did not make it through the PCR process which is why we weren’t able to get and forward or reverse reads for it. Specimen 01 was supposed to be salmon and 03 was mackerel

Lab 05: Marin Fieldtrip!

Allyson Luber

September 30th, 2019

Marin, CA (Mt. Tamalpais State Park), September 24th, 2019

Goal/objective: Observe more populations of Mimulus guttatus in varied habitats around Mt. Tamalpais state park

First stop: Roadside along Highway 1! Beautiful Malibu/SoCal-like views overlooking blue waters and if you’re lucky you’ll catch Marin residents driving their super cars (and you might see some whales in the water)

On the roadside stop of Highway 1 there is natural (safe-to-drink) spring water sourced from Mt. Tam

Next to the spring water spouts is our friends– mimulus guttatus! These flowering mimuli love to get their feet wet and so they all stick around near the water spout. We discussed techniques of how bees pollinate these specific individuals since they’re planted right beside a mountain. Bees would probably fly upwards to find other flowers and plants. Along with mimuli, other water-loving plants surrounded the spring water spouts in a co-habitat, college roommates-living style. These other plants included watercress and horsetails, and we spotted fennel on the other side of the road where there’s dry soil (along the seacoast)

Fresh mint, another one of the plants living in the same habitat as mimulus guttatus in the creek area

Stop #2: Went to lower elevation where the creeks are to find mimuli in different habitats. This habitat was much more shady, cooler temperature, and much more plant/tree life surrounding us

This was one of the populations we spotted of mimuli that was in the creekside area. These mimuli are also dependent on rainfall in terms of flowering timing. For example, a sync population won’t flower because it would be too late in the year and rainfall will soon come. Rainfall is important in the sense that it determines the next generation, not just for mimuli but many of the other plants in this biome. Especially these days with higher variable climate, if there’s a drought year it will potentially take a longer time to flower.

 

Lab 04: Gel Electrophoresis/ ExoSap PCR Clean-up

Allyson Luber

September 17th, 2019

I. Electrophoresis of PCR products

  1. We took out our PCR tubes and let them thaw
  2. After thawing, one of our group members dotted out 13 loading dye dots (~1μl) on a sheet of parafilm (our group did 13 dots compared to the normal 16 dots because there’s only 3 of us)
  3. Next we pipettted 3 μl of each PCR product into its own dot, using a 10 μl pipette
  4. Loaded all dots into the gel (set pipette to 5 μl)
  5. Ran the gel at 130 volts for 30 minutes

Order of lanes on Gel Electrophoresis plate:

(Top)

  1. Negative control
  2. RG- 01
  3. RG-02
  4. RG- 04
  5. EVR-01
  6. EVR-02
  7. EVR-03
  8. EVR-04
  9. AEL-01
  10. AEL-02
  11. AEL-03
  12. AEL-04

15. Ladder

II. Clean-Up of PCR products for sequencing- ExoSAP

  1. Labeled new 0.2 μl PCR tubes with each of my sample codes (label twice on the side of tube)
  2. Make the ExoSAP Master mix – one per table
  • The volume of the Mater mix was calculated based off how many PCR clean-ups our table did
  • Put reagents on ice
  • Pipetted 7.5 μl of each PCR product into a clean, labeled 0.2 μl PCR tube
  • Made the the ExoSAP master mix, keeping the reagents on ice while it was made
  • Pipetted 12.5 μl of master mix into each PCR product tube
  • Placed the tubes in thermocycler and started the ExoSAP program
  • After program was complete, the PCR tubes were placed in a labeled tube rack and into the freezer

Master Mix volumes:

Master mix:                                         Rxn: 1                           Rxns: 13

  1. H2O                                           10.59 μl                            138 μl
  2. 10x buffer (SAP 10x)                 1.25 μl                              16.3 μl
  3. SAP                                            0.44 μl                              5.7 μl
  4. Exo                                             0.22 μl                              2.9 μl

Master mix total:                                 12.5 μl                            162.9 μl

PCR Product

PCR                                                    7.5 μl

Total Cleaned-up volume                    20.0 μl

End result of fish PCR 

(Last 4 lanes to the right, before the ladder, are mine)

Lab 03: Mount Tamalpais

September 10th, 2019

Location: Rock Spring, Mt. Tamalpais, CA.

Plant species observed: Mimulus guttatus

Rock species observed: Serpentine

Background info:

  • Mimulus guttatus is the 2nd most studied plant after arabidopsis
  • Mimulus are edible!
  • Mimulus have a unique “goo” to the leaves
  • Mimulus are diverse because they have great environmental variability and morphology
  • If you can’t distinguish a mimulus plant just remember that their leaves are opposite of each other
  • Like mentioned, mimulus have great temperature variability.
    • They can be found in shady, moist areas along the creek but also in dry, hot spots in open fields.
    • Mimulus have unique interactions with specific environments which makes them adapt to the different habitats
  • In the wet areas along the creek, it’s hypothesized that mimulus get pollinated via water supply and in the drier areas by animal or wind pollination
    • Since mimulus can range over large areas, breeding may be difficult due to different flowering timing
  • Annual plants: only live for one growing season and then die off (like our dry mimulus)
  • Perennial plants: grow year after year after remaining dormant in winter; habitat for maintaining health of plants is important (like our wet mimulus near water because they need moisture to “keep feet wet”).
  • Serpentine rocks: A rock composed of a group of minerals, usually green in color. Some metals found in the rocks are nickel, magnesium, or iron.
    • Tend to get lots of fissures, creating hot springs

Observations

Group of Mimulus guttatus individuals near creek side

An individual dried mimulus guttatus found in the dry, open fields

Serpentine rocks

Views of the city from an area surrounded with serpentine rocks

Lab 02: The Sushi Test

Molecular Ecology Lab 02: The Sushi Test

Allyson Luber — 9/9/19

  • Date, time, and location of purchase of sushi: Monday, September 2, 2019 at 6:52 PM at Sushi House restaurant (Alameda, CA)
  • Fish was stored in separate screw-top mL tubes in refrigerator overnight until lab the next day
  • Group name: Triple Threat
  • Samples (4)
    • Salmon (AEL-01) – bottom right
    • Mackerel (AEL-02) – bottom left
    • Ahi tuna (AEL-03) – top right
    • White tuna (AEL-04)-top left
Sushi sampler

 

Protocol for DNA extraction from animal tissue 

Reagents: DNA extraction kit (Sigma REDExtract-N-Amp Tissue PCR Kit)

  • Extraction Solution (labeled ES)
  • Tissue Preparation Solution (TPS)
  • Neutralizing Solution (NS)

Materials:

  • p200 microcentrifuge
  • 1.5 ml microcentrifuge tubes
  • Razor blades, scissors, scalpels, tweezers
  • Heat block
  • Vortex
  • Ice
  • Sharpie
  • Gloves
  • Paper plate
1. First we individually recorded the information about our samples on the “Animal Tissue DNA Extraction” data sheet, giving each of the samples a unique ID code (code is written next to species name above)
2. We used gloves for protection against the potentially harmful solutions and so our human DNA wouldn’t contaminate the samples.
3. Next we labeled one 1.5 ml Screw-cap or Locking Lid microcentrifuge tube for each of our samples
with the unique ID code. Using a sharpie and writing the unique ID code BOTH on the side and on the
top of the tube to easily identify our samples.
4. Using a razor blade and tweezers, we cut our samples into smaller pieces on a paper plate as well as brushing away debris from the fish, making it pure and clean as possible (from rice or seaweed).
5. With a tare paper we weighed our sample on the scale. The goal is to get 2 – 10 mg of sample tissue, which will be a very small piece.
6. Added 100 μL of Extraction Solution (ES) to each of our labeled sample tubes (use a p200 μl
micropipette and unfiltered tips).
7. Added 25 μL of Tissue Preparation Solution (TPS) to the microcentrifuge tubes with the 100 μl
of Extraction Solution (ES) and micropipette up and down to mix (use a p200 μl micropipette
and unfiltered tips).
8. Carefully, we added each sample to its corresponding extraction microcentrifuge tube using forceps.
9. Take a disposable non-filtered pipette tip and, with our hand we used it to
gently mash our tissue sample up a bit for approximately 2 minutes.
10. Incubated the sample at room temperature for 10 minutes.
11. Moved our samples to the heat block. Incubated the sample at 95 degrees Celsius for 3 minutes.
12. After 3 minutes we took the sample out of the heat block and added 100 μl of Neutralizing Solution (NS) (using a p200 pipette and filtered tips) and mixed by vortexing.
13. Last step was to put samples on ice bucket.
Procedure for Amplifying C01 from Fish
* Use filter tips for all PCR steps*
1. Diluting your gDNA
  • The first step to making a 10x dilution of gDNA was to label a microcentrifuge tube on the top and side with “1:10” including the unique code to avoid confusion later.
  • Added 18 μl of purified water to the tube
  • Added 2 μl of our gDNA to the tube
  • After adding the liquids, I flocked the tube with finger to mix the solution

2. The PCR Reaction

* Just one master mix was made per table *

Master Mix (original volume was multiplied by 13 in order to get master volume, excluding tissue extract)

Reagents

  • Water (PCR Quality): 83.2 μl
  • REDExtract-N-Amp PCR rx mix: 130 μl
  • Forward primer: 10.4 μl
  • Reverse primer: 10.64 μl
  • Tissue Extract (gDNA- 1:10 dilution): 2 μl

Total Volume : 234 μl

  • Added 2 μl of the 1:10 dilution of our gDNA to each of our PCR tubes, except the negative control.
  • Lastly we pipetted 18 μl of the master mix into each of our PCR tubes, including the negative control, making sure to change tips between samples.
  • This reaction was left in ice next to the thermocycler until all PCR reactions were set up. then put the PCR tubes (all samples and the negative control) in the thermocycler and start the reaction, which takes between 1.5-2 hours. The PCR reactions were placed in the freezer when the cycling was complete.
Settings for the thermocycler:
  • 94 degrees C – 4 min (initial denaturation)
  • 30 cycles of:
    • 94 degrees C for 30s (denaturing)
    • 52 degrees C for 40s (annealing)
    • 72 degrees C for 1 min (extension)
  • 72 degrees C for 10 min (final extension)
  • 10 degrees C hold

Results

Fish DNA Barcode results

 

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