Lab 08: DNA Extraction (M. Guttatus)

Allyson Luber

10/22/2019

Modified Alexander et. al tube protocol for DNA Extraction

  1. I started off with labeling 3 2.0 mL tubes with my sample code (001- MAPL, 003- RDRK, AEL-CATB)
  2. Added three sterile 3.2-mm stainless steel beads to each tube provided by Professor Paul
  3. Added a small amount of leaf tissue to each tube- we had to be careful to clean any tweezers or other tools used between tubes to avoid cross contamination
  4. Afterwards, we loaded the tubes within a tube rack into the modified reciprocating saw rack and mounted the rack to the saw. Ours was done by Professor Paul for 40 seconds
  5. Once done on the saw we briefly spun down the tubes in the centrifuge for 15-20 seconds to pull plant dust down from the lids
  6. Next thing, we added 434 μL preheated grind buffer to each of our sample tubes
  7. Incubated buffered grindate at 65 degrees celsius for 10 minutes in water bath, mixing the tubes by inversion every 3 minutes
  8. Added 130 μL 3M pH 4.7 potassium acetate, whilst inverting tubes several times then incubating on ice for 5 minutes
  9. Next, we spun the tubes in a centrifuge yet again at maximum force for 20 minutes, at 14,000 or 15,000 rpm for the tubes in a smaller microcentrifuge
  10. Bought coffee in this 20 minute break (you’ll need it).
  11. Labeled new 1.5 mL tubes with sample ID
  12. When finished centrifuging, transferred supernatant to these sterile 1.5mL microcentrifuge tubes– MAKE SURE TO ONLY GET SUPERNATANT AND NO PRECIPITATE
  13. Added 1.5 volumes binding buffer. It was around 600 μL of binding buffer, to be exact
  14. Applied 650 μL of mixture from step 11 to special little tubies, called Epoch spin column tubes and centrifuged for 10 minutes (until all liquid has passed through) at 15,000 rpm in a centrifuge and discarded flow-through in an erlenmeyer flask
  15. Repeat step 14 with the remaining volume from step 13
  16. Washed the DNA bound to the silica membrane by adding 500 μL of 70% EtOH to the column and centrifuge at 15,000 rpm until all liquid has passed to the collection tube (8 minutes). Discard the flow-through
  17. Repeat step 16
  18. After discarding the flow-through from step 17, we centrifuged the columns for an additional 5 min to remove any residual ethanol
  19. Discarded the collection tubes and placed the columns in sterile 1.5 mL microcentrifuge tubes– make sure these tubes are labeled with sample ID and date
  20. Finally, we added 100 μL preheated (65 degrees celsius) pure sterile water to each tube. Let it stand for 5 minutes and then centrifuged for 2 minutes at 15k rpm to elute the DNA

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.