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October 1 – Introduction to Genieous

LAB: TUTORIAL

This week we went through an introduction of Genieous – a commercial program that performs many functions on DNA and protein sequences. We downloaded the program and made an account [free 2 week trial] and started learning how to use the program.

Two of my reverse reactions from the Sushi Test worked but none of the forward ones did so Prof. Paul assigned extra reactions that I could work with instead to learn about the program. I first downloaded all the fish barcode sequences we would need – “Fish_Barcode_Forward.geneious,” “Fish_Barcode_Reverse.geneious,” “Fish_Barcode_Forward_EXTRA.geneious,” and “Fish_Barcode_Reverse_EXTRA.geneious.” I drag and dropped these files into my Fish Barcode folder that I titled “fish barcodes – yu.”

I already knew my forward reactions did not work so the next step was to look at the two (out of four) reverse reactions to see how well they worked. I looked at the various automatically generated column in the reverse reads folder and saw that the HQ% score was only 9.3% for OY-02 and 2.4% for OY-04. Prof. Paul wrote in the handout that HQ scores of 80% or higher are excellent and 10% may still be usable so I tried to look at them but they were mostly unreadable (oops). The peaks ere unevenly spaced and not very even in height like a good read would be.

The sequences from my two reverse runs were not that short but they were pretty messy and uneven so I decided to continue in the tutorial with the extras titled “KJ03_FbcF_H06” for the forward reaction and “KJ03_FbfR_E11” for the reverse reaction. The forward had an HQ% of 92.9% and the reverse was 96.3%.

After looking at some of the features in the Geneious program, the next setp was to actually assemble forward and reverse sequence reads of the same sample so I highlighted the two KJ03 reads (F and R) and selected ‘De novo assembly.’ This created a new file which held the actual assembly. This showed a consensus sequence, sequence traces for each reads (one of which was the reverse compliment of the original sequence. The next thing I did was edit the two strands (like post transcriptional modification?). I did this by deleting any regions of ‘junk,’ making sure to highlight both the strands and the consensus sequence to avoid any frameshift mutations in the sequence. I also deleted any ambiguities along the sequence (N,Y,etc). If I was able to choose the base based off the complimentary strand I did but most of the time it just showed a dash. Finally, I used the Basic Local Alignment Search Tool (BLAST) to see which organisms had the highest matching sequence to mine.

I found that Thunnus alalunga was the most similar to my sequence and Googled it to find the common name – Albacore.

To find polymorphisms, I went back to the BLAST search results and selected 5 different species and made a nucleotide alignment. There were 7 polymorphisms.

EXERCISES 

 

 

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September 24 – Mimulus pt. 2

This week we drove back to Mount Tamalpais and continued to look at different variations of Mimulus gutatus. We left campus around 1pm and we met at a spot on the mountain where there were streams of fresh water coming from pipes on the side of a cliff. The weather was nice – very sunny and hot at the first location, then shady but warm at the second one (Around 85 F).

You can tell from these pictures that the most abundant areas with the plants were in the wet areas from the water coming from the pipes. Last time we went to Mount Tam Prof. Paul told us that Mimulus like to “keep their feet wet” and this was proof of that.

After we looked around at this spot we drove over to a little hiking area that leads to Muir Woods. There weren’t a lot of Mimulus plants here but we did see a couple individuals.

The picture on the right shows an individual that may not survive to the next generation because they seem like they wouldn’t be hit by water that comes from the creek so it may not have the chance to flower. When we were over here we talked a bit about sink populations and census sizes / effective population sizes – (topics we had touched on in class).

Sink populations –> death rates exceed birth rates (leads to eventual loss and maybe extinction)

Census size / effective population size –> number of individuals needed to have a quantity of interest that is the same in the idealized population as in the real population

After hiking and discussing, we went back to campus around 4:30 pm.

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September 17 – Gel Electrophoresis / PCR Cleanup

This week we ran our DNA samples from the Sushi Test on gel electrophoresis and ‘cleaned up’ the PCR reaction using an ExoSap master mix. To begin, I took my samples from the ice that they were previously stored in and thawed it at room temperature. While they were thawing, we (Mikayla) dotted out 19 loading dye dots on a sheet of parafilm- each dot was about one microliter. The protocol said to put 16 dots but since we had a negative control and a ladder on each row we had three more. After prepping the parafilm, each person at my lab bench pipetted three microliters of their PCR product onto each dot, and Prof Paul put in the ladders on the parafilm for us before running the gel. I then pipetted all of our samples into the gel lanes. Tips were switched on the pipet between samples every time to avoid contamination. [The lanes and sample ID’s are shown below]. Finally, we ran the gel at 130V for 30 minutes.

LANE # (L à R) SAMPLE ID #
TOP  
1 OY01
2 OY02
3 OY03
4 OY04
5 MM01
6 MM02
7 MM03
8 MM04
9 KRS1
10 KRS2
11 KRS3
12 KRS4
13 Negative Control
14 EB01
15 Ladder
   
BOTTOM  
16 EB02
17 EB03
18 EB04
19 Ladder

The next step was to clean up our PCR products for future sequencing. At each table two partners shared a row of 8 0.2 microliter PCR tubes (4 each). We label them with our sample codes on the top, front and back to ensure that our sample would not be lost. Next, we made the master mix. Since we had 16 samples of fish DNA we made enough master mix for 18 reactions. [Recipe is shown below]. All the reagents were put on ice and we only took them off ice when we needed to use them. We pipetted 7.5 microliters of each of our PCR products into our new PCR tubes and then pipetted 12.5 microliter of the Master Mix we made into each of our little tubes. We them put them in the thermocycler and started the EXOSAP program.

 

(Prof. Paul did the last step: After 45 minutes of the program, PCR tubes were placed in a labeled tube rack and placed in the freezer)

Master Mix RXN (1) RXNs: 18
H2O 10.59 microliters 190.6 microliters
10x buffer (Sap 10x) 1.25 microliters 22.5 microliters
SAP 0.44 microliters 7.92 microliters
Exo 0.22 microliters 3.96 microliters
     
Master Mix Total 12.5 microliters 225 microliters

 

 

 

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September 10 – Mount Tamalpais

This week for lab we went on a field trip to Mount Tamalpais. We drove there around 1 pm and got back at 5:00 pm. Once we got there we met Alec, Professor Paul’s graduate student and he led us through a trail to a creek. Over there he and Professor Paul taught us about Mimulus gutatus, the plant that they are studying. This plant shows great variability in morphology and where they can survive so they are a great model system to study. They also have a broad distribution, reach maturity quickly and are one of the first plants that had its whole genome sequenced, which makes it a lot easier to study.

When we got to the creek, each of us sampled a part of the plant and put it in a vial containing silica. Silica dries out the plant so that ‘better’ and more clear DNA can be extracted from it. Alec and Professor Paul taught us how to identify the plant as well- it can sometimes form a mat like structure on the floor and it has leaves that grow opposite to each other.

We labeled the vials with the date, location and our initials.

 

After the creek we walked over to a dry patch of land to see a variant of the Mimulus gutatus plant. We saw that unlike the green, lively variant we saw around the creek, the ones in the dry land were tannish brown and obviously very dried out. The seed pods could be broken open and a lot of tiny brown seeds would come out very easily.

 

Next, we walked over to look at Serpentine rock. Serpentine rock is very harsh for plants and includes heavy metals. There is not a lot of plants that can survive on this rock because of the dryness, heavy metals and the lack of soil around it. It has a greenish color with a copper tint and does not look like it would cultivate any plants that tried to survive there.

We looked at the Serpentine rocks at two different spots (across the street from each other).

Lastly, we walked a trail on the side of the mountain to look at another variant of the Mimulus gutatus plant. This one was interesting because even though the trail was all tan and looked dry, once we found a little part of the trail that has moisture, we found the plant they were studying. This one also flowered and had a yellow “monkey face” flower. We could tell that it was probably bee pollinated (pollination syndrome!) because of the color and the morphology- bees cannot see red and do preferentially pollinate yellow plants usually and the morphology included a landing pad- type petal that would be well suited for a bee to land on and gather/ deposit pollen.

 

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September 3 – The Sushi Test

Pre- Lab 

Tuesday, August 27

Last Tuesday, Mikayla and I went to Ginza Sushi on Haight Street and collected samples for lab the following week. We ordered two rolls and sashimi to ensure that we got four different kinds of fish. The first roll, “Bara” contained maguro as the main fish, the other roll “Ginza” had yellowtail and the sashimi bowl included salmon, akami and yellowtail. We ended up not sampling “Ginza” since we already had yellowtail from the sashimi bowl.

To collect the samples we used chopsticks to pick off a piece about the size of an eraser and put it into the tubes that were provided. To avoid contamination (as much as possible), we used new chopsticks and plates each time.

 

Lab 

DNA EXTRACTION FROM ANIMAL TISSUE

Reagents: We will be using 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

[ Reagents and Materials copied from lab protocol document (JP) ]

  1. The first step was to assign unique codes to each fish sample. These codes and the fish they were assigned to were noted on the “Animal Tissue DNA Extraction” data sheet provided.
    • OY-01 –
    • OY-02 –
    • OY-03 –
    • OY-04 –
  2. Next, we got paper plates, drew lines on it to divide it into 4 spaces to keep the fish samples separate and I cut off a little piece of each- making sure to sterilize the scalpel each time I moved from one fish to another with Kim wipes and ethanol. Each square on the plate was labeled with the code and the fish name and gloves were used as well to avoid contamination of the samples.
  3. After the little pieces were cut I weighed each one on the scale to ensure it was around 2mg [they all came out to be a little over 2 mg]. Separate weigh boats were used for each sample and the scale was zero-ed each time a new weigh boat was on it.
  4. Once all the samples were weighed and ready I labeled four screw cap microcentrifuge tubes with the codes from step 1- I labeled the side and the top.
  5. 100 microliters of Extraction Solution (ES) were then pipetted into each tube using a p200 microliter micropipet. The procedure stated to use unfiltered tips but we could not find any that fit the micropipet so we ended up using filtered tips.
  6. Next 25 microliters of Tissue Preparation Solution (TPS) was added to the same tubes using the same p200 microliter pipet (and new tip). To mix, I gently pipetted up and down using that same tip and pipet.
  7. Finally, each sample was put into its designated tube using forceps- the forceps were cleaned before touching each different sample using ethanol.
  8. Next, using a non filtered tip, I gently mashed each sample in the tubes. Different tips were used for each sample.
  9. The samples were incubated at room temperature for 10 minutes.
  10. After incubation, I moved the samples to the heat block. They were incubated at 95o C for 3 minutes.
  11. After that, I added 100 microliters of Neutralizing Solution (NS) using the same p200 pipette and filtered tips. They were then mixed using the vortex machine.
  12. Lastly, they were put on ice and the extra fish that was collected was given to Professor in case the experiment needed to be repeated.

AMPLIFYING CO1 FROM FISH 

** Everything in this part is using filtered tips **

A. Dilute gDNA

  1. I labeled 4 microcentrifuge tubes with the unique code names and “1:10” on the top and sides.
  2. 18 microliters of purified water was added to each tube.
  3. 2 microliters of each fish sample tube [gDNA] was added to each microcentrifuge tube.
  4. Then I gently flicked each tube to mix it.

B. PCR reaction

  1. For the PCR reaction we needed to make a master mix with our lab benches. We had 16 samples total (4 each) so we made a master volume to account for 18 samples so we would have a little extra just in case. The amounts of each reagent are shown below.
    • Reagent                                                            VolumeWater (PCR Quality – autoclaved, filtered)     6.4 mlMaster volume (volume x 18) = 115.2 microliters

      REDExtract-N-Amp PCR rxn mix                  10 ml

      Master volume (volume x 18) = 180 microliters

      Forward Primer FbcF                                       0.8 ml

      Master volume (volume x 18) = 14.4 microliters

      Reverse Primer FbcR                                       0.8 ml

      Master volume (volume x 18) = 14.4 microliters

      Tissue Extract (gDNA) (1:10 dilution)            2 ml

      Total Volume                                                  20ml

      Master volume (volume x 18) = 324 microliters

2. Next, we got PCR tubes and labeled each with the codes from earlier. Everyone got 4 tubes except for one of us who got 5 so we used that extra one as a negative control.

3. 2 microliters of the 1:10 dilution of the gDNA was affix to each of the small PCR tubes except for the negative control. Tips were changed between each sample.

4. Next 18 microliters of the master mix were added to each tube, including the negative control. Again, tips were changed between each sample.

5. This was all put on ice until all the PCR reactions in the lab were ready to go. Then we put them all in the thermocycler for the reaction.

6. Professor Paul ran the reaction – settings are shown below and it took about 1.5 – 2hours for the full reaction. They were then put 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