Objective:

The objective of today’s lab is to finalize our poster presentations for the CURE symposium.

Purpose:

The purpose of today’s lab is to prepare for the CURE symposium.

What did we do?:

Poster Title: Environmental DNA in the Rhizosphere Surrounding Quercus incana

Final Abstract

Both marine and soil ciliates are eukaryotic microorganisms that play an integral part in the health and prosperity of aquatic and terrestrial food chains, respectively. Marine ciliates are often the targets of biodiversity research, but soil ciliates are often overlooked in scientific studies. This field experiment focuses on using metabarcoding to detect the presence of the V4 region of the 18s ribosomal subunit, a genetic sequence unique only to eukaryotic organisms. The soil sample we collected was from a Bluejack Oak, or Quercus incana, near Earle Hall and East Village on Baylor University’s campus. The sample was brought back to the lab to be tested for different types of metadata, such as pH and soil texture. For DNA extraction, we used silica beads to break open the cell membrane of the microorganisms, DNA extraction buffer to pull the DNA from the lysed cells, and activated charcoal powder to remove impurities from the new DNA sample. Once we obtained a purified DNA sample, we performed a polymerase chain reaction to amplify the eDNA. This amplified DNA was used in gel electrophoresis, where we determined both the mass of our DNA and the length of each sequence.  We also performed a nanodrop experiment in which we found the concentration of DNA. Based on our results, we were able to confirm the presence of the 18s ribosomal subunit, thus signifying that ciliates are present in our sample. Our DNA can be sequenced and analyzed in conjunction with our metadata to increase our understanding of soil ciliate biodiversity.

Conclusion:

I fixed some of the organization issues in the poster and added “why does this matter” statements to the intro and the conclusion.

Future Goals:

I hope to have a successful presentation at the symposium.

Objective:

The objective of today’s lab is to analyze the eDNA sequenced last year

Purpose:

The purpose of today’s lab is to analyze the eDNA using qiime2 to practice

Procedure:

1. Check if ITS removed qiime by typing source activate qiime2-2019.1
2. Download the box file on to the mac
3. Import the sequences into qiime as a .qza file
4. Demultiplex the sequences
5. Denoise the sequences using DADA2
6. Create a feature table
7. Create a phylogenic tree
8. Create a taxa bar plot

Conclusion:

It was successful, even if it took really long for the computer to process.

Future Goals:

I hope you get better, have a nice easter, and to use this info later.

Objective:

The objective of today’s lab is to practice qiime 2 through the moving pictures tutorial

Purpose:

The purpose of today’s lab is to learn how to use qiime 2 in order to use it for our own purposes.

Procedure:

1. Ensure conda and qiime 2 is installed in the terminal this can be done by typing conda update conda (to check conda) and source activate qiime2-2019.1
2. Make a new directory named “qiime2-moving-pictures-tutorial” using the mkdir command
3. Change to that directory using the cd command
4. Using the wget command get the sample metadata from the qiime website
   1.  wget \
      -O “sample-metadata.tsv” \
      “https://data.qiime2.org/2019.1/tutorials/moving-pictures/sample_metadata.tsv”
5. Make a new directory named “emp-single-end-sequences”
6. Using the wget command get the sample barcodes and sequences
   1.  wget \
      -O “emp-single-end-sequences/barcodes.fastq.gz” \
      “https://data.qiime2.org/2019.1/tutorials/moving-pictures/emp-single-end-sequences/barcodes.fastq.gz”
   2. wget \
      -O “emp-single-end-sequences/sequences.fastq.gz” \
      “https://data.qiime2.org/2019.1/tutorials/moving-pictures/emp-single-end-sequences/sequences.fastq.gz”
7. Import

Conclusion:

Future Goals:

Objective:

The objective of today’s lab is to install QIIME and practice it.

Purpose:

The purpose of this lab is to install QIIME in order to use it for translating our sequenced data.

Procedure:

1. Install 64 bit.pk for Mac/Os conda from miniconda for your personal use only

2.Close and Open terminal

3.Type conda update conda

4.When prompted to Proceed type y

5. Type conda install wget

6.Copy and paste commands below into terminal

wget https://data.qiime2.org/distro/core/qiime2-2019.1-py36-osx-conda.yml

conda env create -n qiime2-2019.1 –file qiime2-2019.1-py36-osx-conda.yml

7. Type conda activate qiime2-2019.1

Conclusion:

Overall very successful lab, I had no trouble with installing conda even though it was my first time on a mac.

Future Goals:

I hope to use this knowledge for future research opportunities.

Objective:

The objective of today’s lab is to learn about the next generation sequencing

Purpose:

The purpose of today’s lab is to learn next gen sequencing(NGS) as it is the next step of our research

What did we learn:

Illumina Sequencing

1. The first step in this sequencing technique is to break up the DNA into more manageable fragments of around 200 to 600 base pairs.
2. Short sequences of DNA called adaptors, are attached to the DNA fragments.
3. The DNA fragments attached to adaptors are then made single stranded. This is done by incubating the fragments with sodium hydroxide.
4. Once prepared, the DNA fragments are washed across the flowcell. The complementary DNA binds to primers on the surface of the flowcell and DNA that doesn’t attach is washed away.
5. The DNA attached to the flowcell is then replicated to form small clusters of DNA with the same sequence. When sequenced, each cluster of DNA molecules will emit a signal that is strong enough to be detected by a camera.
6. Unlabelled nucleotide bases and DNA polymerase are then added to lengthen and join the strands of DNA attached to the flowcell. This creates ‘bridges’ of double-stranded DNA between the primers on the flowcell surface.
7. The double-stranded DNA is then broken down into single-stranded DNA using heat, leaving several million dense clusters of identical DNA sequences.
8. Primers and fluorescently-labelled terminators (terminators are a version of nucleotide base – A, C, G or T – that stop DNA synthesis) are added to the flowcell.
9. The primer attaches to the DNA being sequenced.
10. The DNA polymerase then binds to the primer and adds the first fluorescently-labelled terminator to the new DNA strand. Once a base has been added no more bases can be added to the strand of DNA until the terminator base is cut from the DNA.
11. Lasers are passed over the flowcell to activate the fluorescent label on the nucleotide base. This fluorescence is detected by a camera and recorded on a computer. Each of the terminator bases (A, C, G and T) give off a different colour.
12. The fluorescently-labelled terminator group is then removed from the first base and the next fluorescently-labelled terminator base can be added alongside. And so the process continues until millions of clusters have been sequenced.

Conclusion:

Today was interesting as we were introduced to NGS, the illumina sequencing method, cloud computing, and QIIME. This was significant as we would be using these resources for our research.

Future Goals:

I hope to use these resources later on in future labs.

Objective:

The objective of today’s lab is to review our poster

Purpose:

The purpose of today’s lab is to review our poster in order to fix it up

Current Presentation:

What We Need to Fix :

• Change the title – we were under the pretense that we would get to go through with the sequencing
• Make a flow chart for methods
• Add tree metadata, nanodrop, and gel electrophoresis (All formatted)
• Fix formatting on the PCR gel
•  In general, add more information to the results

Conclusion:

In summary, there is a lot of problems that need to be solved

Future Goals:

I hope for a successful poster presentation.

Objective:

The objective of today’s lab is to start running PCR on our DNA.

Purpose:

The purpose of today’s lab is to start the last portion of the procedure in metabarcoding.

Procedure:

1. Obtain your 1.5x agarose gel, your DNA sample, your control sample, and DNA ladder template.
2. Place gel into the TAE liquid in the electrophoresis machine
3. Load 10μL of the PCR samples and 5μL of ladder into the wells
4. Electrify the samples at 100v for 30mins

Observations:

Above is our gel the DNA samples are in (From left to right) wells 3 and 4. Thankfully we found DNA fragments on the V4 segment.

Storage:

All samples were returned to the cooler and the gel was still being electrified when I left for the computer lab so I assume that either Dr. Adair or someone else properly stored it.

Conclusion:

The lab was successful as we have some DNA fragments on the 18s.

Future Goals:

I hope everyone has a wonderful spring break.

Objective:

The objective of today’s lab is to prepare our DNA for PCR.

Purpose:

The purpose of today’s lab is to prepare our DNA to be amplified through PCR.

Procedure:

1. Calculate the amount of DNA to be used in ng/µl and if it needs to be diluted
2. Obtain two PCR tubes
3. Fill the first PCR tube with the DNA, the master mix, primers, and water ADD WATER LAST
4. Fill the second PCR tube with just the master mix, primers, and water this is control
5. Store the PCR tube and the control tube on a rack for thermocycling.

Observations:

Above is the calculated amounts of the Extracted DNA Solution. Prior to adding the amounts the table was sterilized with bleach in order to improve our chances of amplifying only our DNA. We didn’t need to dilute the DNA as there was enough DNA to gain 1µL in the extracted DNA.

Storage:

The control tube and the extracted DNA tube was stored in columns E3 and E4

Conclusion:

The lab was successful we had a small hiccup with the PCR tube as we accidently put the contents in the wrong tube, but we fixed it by getting the correct tube.

Future Goals:

I hope that my group’s DNA is the one to be sequenced and that the test doesn’t kill me.

Objective:

The objective of today’s lab is to analyze the recently purified DNA in gel electrophoresis.

Purpose:

The purpose of today’s lab to finally observe the DNA that we have been preparing for weeks.

Procedure:

1. Practice filling wells with 10x buffer liquid
2. Obtain gel and purified DNA
3. Break gel out of its mold and place in gel electrophoresis machine
4. Add the 10µL DNA samples and 5µL DNA mass standards into individual wells
5. Turn on the gel electrophoresis and run 100 volts for 20 minutes
6. Take gel up to a machine to be analyzed

Observations:

The contents of the wells were in this order: Well 2 has Group 1’s DNA, well 4 had the low DNA Mass Standard, well 5 had the high DNA Mass Standard, Well 7 had Group 3’s DNA, and all other wells were used for practice.

Storage:

The gel was given to LA.

Conclusion:

The lab was successful for both groups as both DNA samples showed high amounts of DNA, and don’t have to repurify DNA

Future Goals:

I am excited to do PCR next week and hope for positive results.

Objective:

The objective of today’s lab is to purify the soil DNA and prepare the gel to be use for gel electrophoresis.

Purpose:

The purpose of today’s lab is to continue making progress on DNA sequencing.

Procedure:

DNA Purification

1. Obtain the “Crude soil DNA” and transfer it to a conical tube it should be 1mL, but if not add D.I Water
2. Add 2mL of warm DNA resin to the “Crude soil DNA” and mix together by inverting the tube multiple times
3. Obtain the syringe barrel and column, assemble together and attach to the vacuum filitration manifold
4. Transfer half of the contents of the tube into the syringe barrel and allow it to seep entirely through before transferring the other half  into the syringe
5. Wash the interior of the syringe barrel with 2mL of 80% Isoprophyl alcohol 6 times to ensure all the DNA from the soil DNA and DNA resin mixture is caught in the column
6. Detach syringe from vacuum filitration manifold and transfer the contents of the column to an Eppendorf tube
7. Spin tube in a centrifuge for 1 minute
8. Incubate for 1 minute
9. Spin again for 1 minute
10. Label and store the recently spun tube in the freezer

Agarose Gel Creation

1. Fill a conical tube with 4mL of stock solution and 36mL of D.I. water
2. Add 0.4g of agar powder and the stock-D.I. water solution to an Erlenmeyer flask
3. Swirl gently and microwave for 1 minute( it should be clear as water after heating)
4. After cooling the solution off, have either the TA or LA to add 2mL of EtBr to the solution and swirl together
5. Obtain gel electrophoresis mold
6. Pour the solution into the mold and wait for it to cool
7. Obtain sandwich bag and fill the mold with 10x TAE buffer solution
8. Label and store the mold in the freezer

Observations:

Austin and I did the DNA purification and Emely did the Agarose gel.

Storage:

The purified DNA tube and the gel mold are both stored in the freezer.

Conclusion:

This was a very successful lab eventhough I think that was the longest lab this semester and it was exciting to learn how DNA is purified and how to make agarose gel.

Future Goals:

I am excited to start doing PCR on our purified DNA and discover what microorganisms live in the soil on campus.