Sydney Ortenberg

April 26, 2019

Lab 14: Abstract and Poster

Purpose:

The purpose of this lab was to improve our abstracts and posters for the Cili-CURE symposium. We also needed to vote on a logo and enter our information into the dropbox and class spreadsheets.

Procedures:

1. vote on logo
2. write down identifier on test tube
3. record metadata on shared excel sheet
4. work on abstract and poster
5. upload abstract and poster to Dropbox

Data:

Soil ID: LOS22_3Sp19

GPS Location: -97, 30

Tree Species: Quercus Virginiana

BHD: 140cm

pH: 6.0

Soil Texture: sandy loam

Extraction: Silica Bead

DNA Concentration: 96ng/uL

Volume: 500

PCR: positive

Soil Label: SLO s2019

Abstract:

Ciliates play a major role in the food web which impacts all hierarchies of life. There is vast biodiversity in the rhizosphere of Earth, containing ciliates and other microorganisms (1). Little is known about the abundance of organisms that reside in the soil of terrestrial environments. This study was conducted to determine the diversity of soil ciliates in the rhizosphere on Baylor University’s campus. The process included collection, extraction, and amplification of ciliate DNA from the soil samples (2). The sample was retrieved from a Quercus Virginiana tree on Baylor’s campus in sandy loam soil. Our PCR for the 18S V4 primer resulted in a positive DNA band although the DNA concentration was not completely pure. Our results indicate that there is a large concentration of ciliates in the rhizosphere of trees on Baylor University’s campus.

Title: Investigating Soil Ciliate Biodiversity

Storage: Data tubes were placed back on the rack.

Conclusion and Future Steps:

This lab allowed us to wrap up some things before we have our presentations and the symposium. We needed to record all of our data on the class spreadsheet so it is all organized and all of the data is in one place. We also took feedback from our TA and LA’s to improve our abstract and poster so they can be printed. As a group, we all edited our abstract and poster to make it better. We also voted on a logo for a sticker or T-shirt so we can always represent Cili-CURE throughout Baylor. In the future, the posters will be printed and we will have the CURE symposium in which we will show our posters and also have a presentation in class.

Sydney Ortenberg

April 19, 2019

Lab 13: Soil eDNA and Qiime2 Analysis

Purpose/Objective:

The objective of this lab was to go through all the code and successfully download it to our computers. The purpose of this lab was to use qiime once again to understand its functions and obtain the visualizations from it. The data we used was all of the data that was collected from cili-cure the previous year.

Procedures:

1. ensure that qiime 2 is downloaded on computer and if not, redownload it.
2. Download the folder in the powerpoint from the module.
3. Activate QIIME 2 in the terminal by typing “source activate qiime2-2019.1
4. use the walkthrough document and copy and paste all the code as listed below:

Importing sequences as QIIME2 artifact:

qiime tools import \

–type EMPPairedEndSequences \

–input-path emp-paired-end-sequences \

–output-path emp-paired-end-sequences.qza

Demultiplexing sequences:

qiime demux emp-paired \

–m-barcodes-file sample-metadata.tsv \

–m-barcodes-column BarcodeSequence \

–i-seqs emp-paired-end-sequences.qza \

–o-per-sample-sequences demux.qza \

qiime demux summarize \

–i-data demux.qza \

–o-visualization demux.qzv

Denoising using DADA2 and creating a feature table with Representative sequences:

qiime dada2 denoise-paired \

–i-demultiplexed-seqs demux.qza \

–p-trunc-len-f 220 \

–p-trunc-len-r 220 \

–o-table table.qza \

–o-representative-sequences rep-seqs.qza \

–o-denoising-stats denoising-stats.qza

qiime feature-table summarize \

–i-table table.qza \

–o-visualization table.qzv \

–m-sample-metadata-file sample-metadata.tsv

qiime feature-table tabulate-seqs \

–i-data rep-seqs.qza \

–o-visualization rep-seqs.qzv

qiime metadata tabulate \

–m-input-file denoising-stats.qza \

–o-visualization denoising-stats.qzv

Creating a phylogenetic tree:

qiime phylogeny align-to-tree-mafft-fasttree \

–i-sequences rep-seqs.qza \

–o-alignment aligned-rep-seqs.qza \

–o-masked-alignment masked-aligned-rep-seqs.qza \

–o-tree unrooted-tree.qza \

–o-rooted-tree rooted-tree.qza

Taxonomic classification:

qiime feature-classifier classify-sklearn –i-classifier silva-132-99-515-806-nb-classifier.qza –i-reads rep-seqs.qza –o-classification taxonomy.qza

qiime metadata tabulate \

–m-input-file taxonomy.qza \

–o-visualization taxonomy.qzv

qiime taxa barplot \

–i-table table.qza \

–i-taxonomy taxonomy.qza \

–m-metadata-file sample-metadata.tsv \

–o-visualization taxa-bar-plots.qzv

**Note: Any time a .qzv was entered, the file was dragged and dropped into the view qiime 2 website.

Data:

Storage: 

N/A since in computer lab

Conclusion and Future Steps:

The purpose of this lab was to use qiime once again and grasp more knowledge of it before we sequence our own DNA. We went through several steps and we were able to make a phylogenetic tree and compare the powersoil eDNA and the Chelex eDNA. We were able to see the abundance of the different soil types and use the visualizations to understand the data. In the future, I am looking forward to using qiime to metabarcode our findings and us the databse to compare it to other V4 regions.

Sydney Ortenberg

April 5, 2019

Lab 11: Cloud Computing (Qiime)

Objective/Purpose:
The objective of this lab was to install Qiime and learn some of its commands. The purpose was to start learning the process of coding and metagenomics.

Procedures:

1. Access terminal page from computer
2. Download Miniconda 64-bit (.pkg installer) to computer
3. Once download, install for your personal use only
4. Close terminal
5. Reopen terminal
6. Type ‘conda update conda’ and allow to process
7. Type ‘wget https://data.qiime2.org/distro/core/qiime2-2019.1-py36-osx-conda.ym conda env create -n qiime2-2019.1 –file qiime2-2019.1-py36-osx-conda.yml”
8. allow computer to process and finish the job

List of commands Learned:

• y (yes): How to proceed with a command
• say: tells computer to say something using the speaker
• conda update conda: updates miniconda
• conda install wget: installs widget on miniconda

Data: N/A since all completed on computer and it disappeared

Storage: N/A since in computer lab

Conclusion and Future Steps:

In conclusion, this lab was very beneficial and I think we learned a lot. Qiime will allow us to analyze large samples of DNA and compare it to each other. Overall Cyverse is a large platform that allows researchers to compute large samples of data. Requesting access to the atmosphere was an important step so we can get allowed in and start computing our metagenomics. It was an enjoyable lab and I am looking forward to completing our metagenomics soon.

Sydney Ortenberg

March 29, 2019

Lab 1o: Next Generation Sequencing & Metabarcoding

Objective/Purpose:

The objective of this lab was to introduce another type of sequencing; Next Generation Sequencing and Illumina. The purpose of this lab was to learn about other types of sequencing, what it does, and also learn about a large database for metagenomics called CyVerse.

Procedures:

1. Edit Poster prior to lab to turn in for final draft.
2. Assort Illumina steps in order using the playing cards with a partner.
3. Create a username for Cyverse and record on the class spreadsheet.
4. Complete QTM.

Data:

The chronological steps of Illumina sequencing:

1. Break up genomic DNA into more manageable fragments of around 200 to 600 base pairs by using enzymes or by PCR.
2. Tag the DNA fragments with short sequences of DNA called adaptors.
3. Denature the double-stranded molecules into single-stranded molecules that have the adaptor and primers binding site. This is done by incubating the fragments with sodium hydroxide.
4. Attach the DNA fragments to the flow cell through complementary binding of the adaptors to the oligos (primers) on the surface of the flow cell.
5. Replicate the DNA attached to the lyocell to form small clusters of DNA with the same sequence.
6. Unlabeled nucleotide bases and DNA polymerase are then added to lengthen and join the strands of DNA attached to the flow cell. This creates ‘bridges’ of double-stranded DNA between the primers on the flow cell 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 labeled terminators (a version of nucleotide base- A, C, G, or T- that stop DNA synthesis) are added to the flow cell.
9. The primer attaches to the DNA being sequenced.
10. The DNA polymerase then binds to the primer and adds the first fluorescently-labeled 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 flow cell 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 color.
12. The fluorescently-labeled terminator group is removed from the first base and the next fluorescently-labeled terminator base can be added alongside. And so, the process continues until millions of clusters have been sequenced.

Storage: N/A since in computer lab.

Conclusion/ Future Steps: 

This lab introduced us to a different sequencing method called Next Generation Sequencing and we were able to become familiar with its methods so we can use it. Through the presentation of the history and sorting out the cards, we were able to see how the process worked. The Cyverse database allows scientists and researchers to analyze large amounts of data since most normal computers do not have the capacity to do so. In the future, I am looking forward to using Cyverse to analyze our data from this past semester and see how it all plays out. I think that Cyverse is a unique platform and we can learn a lot from it.

Sydney Ortenberg

March 22, 2019

Lab 9: Poster Presentations

Objective/Purpose:

The objective of this lab was to present our rough draft posters that we created the previous week. The purpose of this was to get critiqued on it and see what we needed to do. We were also able to see other groups and learn more about what a poster is supposed to look like. This lab helped us improve our poster for the final presentation, and also allowed us to become more comfortable presenting in front of audiences.

Procedures:

1. present poster
2. critique others
3. asses what needs to be done to your poster

Data:

Storage: N/A

Conclusion/Future Goals:

In conclusion, this lab was very helpful in that it allowed us to see what our poster needs to have on it and what we need to do to fix it. We compiled a list of everything we need to add/modify which is in the picture above. I was able to see what a good scientific poster should have on it. In the future, I will work on modifying my poster so it is better, and I also hope to create a good, aesthetically pleasing, scientific poster.

Sydney Ortenberg

March 8, 2019

Lab 8: PCR Results

Objective/Purpose:

The objective of this lab was to perform PCR gel electrophoresis and begin planning out our poster. The purpose of this lab was to see our DNA samples under the PCR electrophoresis and determine whether or not our sample meets the qualifications to be sequenced.

Procedures

It is important to Wear gloves during this lab because there are caustic items.

1. Obtain the 1.5% agarose electrophoresis gel prepared last lab.
2. Obtain the 1 kb ladder sample and the control and treatment DNA samples that were prepared last lab.
3. Add 5ul of the 1kb ladder into the first lane.
4. Add 10ul of each sample into their designated lanes.
5. Run the gel for 30 minutes at 100V.
6. Dr. Adair took the gels to the secondary lab and analyzed them with the MCB C305.

Data:

Storage:

The gels were left in the fridge at 4 degrees Celcius. All equipment was disposed of and cleaned up.

Conclusion and Future Steps:

In conclusion, this lab allowed us to perform PCR electrophoresis in order to analyze our DNA to see if it is worthy of being sequenced by a company. If it is up to standard, it will be sent off to be sequenced and then shared with the whole scientific community. The midterm quiz allowed us to reflect everything we have learned this year and test to see whether we have paid attention to details and grasped everything we have done. The brainstorming of our poster allowed us to start thinking about how we are going to present everything and have an overall idea of what we are trying to accomplish. I am excited to analyze my DNA and see whether it can be sequenced or not. I am also looking forward to making our poster because I think it will be a unique and creative way to present all of our findings.

Sydney Ortenberg

March 1, 2019

Lab 7: PCR Amplification

Objective/Purpose:

The objective of this lab was to modify our DNA sample in order to be used for PCR in next weeks lab. The purpose of this lab was to create our DNA for PCR so we can meta barcode our ciliate DNA to be used for the scientific community.

Procedures:

1. Wear gloves and clean the desk and work area with 10% bleach to sterilize the environment.
2. Place an empty tube, water, DNA sample, and V4 Primer in an ice bath.
3. Dilute DNA Sample if necessary  (in our case, we didn’t have to)
4. Take two tubes which have already containing 12.5ul 2X Taq Mix (one for control and one for treatment)
5. For the control tube, add 1ul primer and 11.5ul water and centrifuge for 15 seconds.
6. For the treatment tube, add 1ul DNA sample, 1ul primers, 10.5ul water and centrifuge for 15 seconds.
7. place in tube rack with other classes’ and label where you placed your tube on the sheet of paper.

Data:

Storage:

The ice bath and its contents of primer and water were taken back to the right area. The newly created DNA tubes were placed in the class storage rack and labeled where our group was placed. All other scientific equipment was disposed of and put back into the correct spot.

Conclusion and Future Steps:

Overall, this lab was successful and my group created this new DNA sample with ease. My group was lucky in that we did not have to dilute our DNA sample because our concentration was 96.3nm and that was close enough to 100ng. My group and I paid attention to small details so there would not be any mistakes or contamination of our DNA amplification tubes. I am looking forward to performing the PCR and what kind of results we get. It will be interesting to see what information we can take from our DNA sample and how far this whole experiment has come. I am thinking about what our poster will look like and how we will design it, and I am also starting to prepare for my midterm exam.

Sydney Ortenberg

Feb 22, 2019

Lab 6: Running the Gel

Objective/Purpose:

The objective of this lab was to run the gel electrophoresis and analyze our DNA within the gel. The purpose of this lab was to hopefully find DNA within our sample. The nanodrop technology would analyze our sample and see how pure it is and if there was any contamination.

Procedures:

Gel Electrophoresis

1. Remove the gel from the mold and transfer it onto the transfer dock and cover the gel and the rest of the docker with 1X TAE buffer (with gloves on)
2. Select the walls in which the DNA, mass standards and practice buffer will be put in.
3. Extract 9 uL of DNA and transfer it into Eppendorf tube
4. Add 1 uL of 10X loading buffer to the Eppendorf tube
5. Mix solution with the vortex for 1o seconds.
6. Add 10 uL of DNA from your DNA sample into a well
7. Add 5 uL of DNA 31 ng mass to a well
8. Add 5 uL of DNA 500 ng mass to a well
9. Add 10 uL of DNA from the other group’s sample to different well
10. Run the agarose gel at 100 V for 20 minutes
11. Take the gel to the UV illuminator and observe the image to see how much DNA was found in your sample by comparing it to the mass standards.

Nanodrop Technology:

1. Clean the machine and ensure the machine is clean and dry. Neutralize the machine by placing a drop of water on the machine first, and running it.
2. Place 1 uL of DNA sample on the top of the holder
3. Close the machine and wait for results
4. Analyze the results that appear on the screen.

Data and Results:

• we do not have access to the images of the Gel under the UV light

Storage:

The gel mold was disposed of and all other equipment was stored away. The gel mold container was bleached and cleaned and put in the correct area.

Conclusion and Future Goals:

In conclusion, we were able to detect DNA in our sample from gel electrophoresis. The agarose gel showed us the different sizes of our DNA bands compared to our mass standards, and we were able to view them. More analysis will take place that will allow us to identify our band sizes more specifically. We were also able to use a Nanodrop spectrophotometer to gather data relating to the concentration and purity of DNA present in our sample. Using this, we found that our DNA contained some impurities. DNA should be at 1.8 and our sample was 1.61. In the future, we will be figuring our DNA bands more exactly, and comparing them to others, and we will also be sending them off to be analyzed and grouped together through bioinformatics.

Sydney Ortenberg

Feb. 14. 2019

Lab 5: DNA Purification and Agarose Gel

Purpose/Objectives:

The objectives of this lab were to purify our DNA extracts from last lab and create our agarose gel. We will use the agarose gel to run gel electrophoresis and ultimately sequence our DNA.  The purpose of this lab was to create the items we need in order to sequence our DNA and discover the biodiversity of ciliates.

Procedures:

DNA Purification

1. The “crude soil DNA” extraction was obtained and pipetted into a 15 mL falcon tube.
2. 2 ml of warm DNA resin (at 37 degrees) was pipetted into the tube.
3. A column bottom on a syringe barrel was set up on a vacuum filtration manifold.
4. Half of the DNA/resin mixture was added to the column.
5. After all of the liquid was filtered through, the other half of the DNA/resin mixture was added slowly to prevent any clumps.
6. After all the remaining liquid was pulled through, 2 ml of 80% isopropanol was added and filtered. This was repeated two more times for a total of 6 mL.
7. The column was removed and placed in a 1.5 mL tube and was centrifuged for five minutes.
8. The column was removed from the tube and placed into an 80-degree incubator for one minute.
9. The column was then placed into a new 1.5 ml tube and 50 ul of sterile heated water was added to the column.
10. The column was incubated for an additional minute before it was taken out and re-spun for one minute.
11. The tube was labeled and placed into the freezer at -20 degrees.

Agarose Gel

1. 4 mL of 10x Tris-EDTA-Acetic acid stock (TAE) was added into a conical tube with 36 mL of DI water to create the stock solution.
2. 0.4 g of agarose was weighed out and added to an Erlenmeyer flask.
3. The 40 mL of the stock solution was added to the Erlenmeyer flask.
4. The flask was microwaved for 45 seconds (or until it fully dissolved) before 2mg of Ethidium Bromide was added to it.
5. The gel mold was assembled, and the stock solution was added.
6. After 30 minutes, the solution had solidified, the grate was taken out and enough 1x TAE was added to completely cover the gel.
7. The mold was labeled, placed in a bag, and placed into the fridge until next lab.

Storage:

All caustic items were placed in the biohazard trashcan, and all scales, pipettes, and tubes were cleaned or disposed of. The DNA purification tube was placed in the freezer and the gel mold was placed in the refrigerator.

Conclusion/Future Steps:

This lab taught us the importance of purifying our DNA and removing impurities that were in the sample. Learning how to create the gel was also important so we can run electrophoresis on our DNA sample. This lab taught us patience and that the smallest steps matter in creating the overall big picture. I am looking forward to sending our DNA samples off and using bioinformatics to see the results.