GPS: N, W. Physical Description: Air Temperature: 78 F. Collection Date: First bacteria we worked with- 08/29/17,

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1 Annie Choudhry Research habits of the mind- for your phage lab workout during DNA prep Hyperlink a file using the hyperlink button Phage name TuttiFruity Soil Sample Collection GPS: N, W Physical Description: Small sample of soil from near the coastline Air Temperature: 78 F Collection Date: First bacteria we worked with- 08/29/17, Cove Bacteria September 2016 Discovery method Enrichment plating choose: Direct or Enrichment plating Identification of plaque Date: Appearance: cloudy Purification of plaque 3 rounds of Purification (Number and method used) Describe final plaque morphology cloudy around edges and clear center (3-4mm) (Clear/turbid/halo/comet/size/multiple, etc.) Conditions for production of web plate suitable conditions Collection of high-titer lysate Titer: 10^8 pfu/ml DNA purification, how many columns did you use? 2 Date:

2 DNA quantification, Concentration/volume: Concentration: Estimated volume collected: How is your tube labeled? Restriction digest, which enzymes cut? EcoRi, Haell, uncut EM Visualization Head Diameter: Tail Length: Submitted Archive Report to bacillus.phagesdb.org or phagesdb.org (Microbacterium phages) Submitted lysate archive to Dr. Johnson 2017VACVTuttiFruityZA How is your tube labeled? file was unable to upload that day Took our soil sample today and inserted 1 ml of solution through a syringe and filter into the small tubes. Everything was near flame for sanitization purposes. Then through the pipette 1 mil of the filtered soil solution was then placed on the plate of agar completely covering it's surface. Oluchi Chigbu 8/31/ The filtered soil solution that was placed on the agar last class was today found to have a phage. Therefore we took our individual filtered soil solution and did a series filtration from 10^0 to 10^-5 after placing 90mL of phage buffer into each microtube. Then 10uL of the filtered microtubes was placed into the test tubes of microbacterium foliorum. Time period of 15 min passed. Agar was added to the test tubes with microbacterium foliorum and then plated. A total of 6 plates were used.

3 Oluchi Chigbu 9/5/ The 6 plates today today were found to be contaminated. Therefore the same procedures as the day before were done. 9uL of phage buffer were added into each microtube labeled from 10^-1 to 10^-5. Then 10uL of the filtered soil solution was added to 10^-1 and so on. A series dilution was done. The test tube containing micro-bacterium foliorum had top agar placed into it and poured on to the petri dish. The petri dish was labeled and divided into six sections. Then a sample of 10uL from each dilution was spotted into it's rightful section. A spot test was done. Rahul Warrier 9/7/17 9/12/17 Take a conical tube and pour 25mL of bacillus media into the tube. Then pipette 0.5mL of bacillus thuringiensis into the tube of media. Take your soil and place it into the tube till it reaches the measurement of 35mL. All procedures were done under flame for sanitation purposes so no contamination would occur. The tube will now rest in a cool place and chill. Ayshah Asmat 9/12/ Today I performed another filtration of my soil sample. The filtration happened by first injecting the soil sample into the syringe through the pipette. Then transferring it from the syringe though the sterile filter into the small tube. Our petri dish was the bacillus. The mixed solution was bacillus and top agar was placed in the petri dish. Then the filtered sample solution was spot tested. Ouch Chigbu 9/19/ A phage was found using the bacillus bacteria. The pipette was dipped into the phage and back into the small tube containing 100mL of phage buffer. Five minutes passed and 90 ml of phage buffer was added to every small tube labeled from 10^-1 to 10^-5. While pipetting it was made sure a new tip was exchanged for every other dilution, and every time phage buffer was added to the small tube. Then a series dilution was done from 10^0 to 10^-1. Top agar was added to the petri dish and we waited till the top agar set. Then we pippetted each series dilution from 10^0 to 10^-1 onto the petri dish. This was all done under flame for contamination purposes. Rahul Warrier There was no phage found on petri dish due to the absence of adding bacillus bacteria to the top agar. Therefore the steps were repeated from last class ^^^^. A lawn was made by adding bacillus bacteria to TSB top agar and plating it. Then we conducted a series dilution with the small tube containing the phage bacteria. All six portion from 10^0 to 10^-5 were plated using pipettes. Then we wait again for next class. Oluchi Chigbu 9/28/17

4 The filtrated bacillus phage with the bacteria was taken to conduct a series dilution to 10^-5. Each small tube from 10^-1 to 10^-5 contained 90ml of phage buffer and while conducting the series dilution 10ml is transfered. Each dilution has it's own plate with a total of 6 plates. On each plate a mixture of bacillus bacteria and TSP top agar were poured on the plate. Then 10ml of each dilution was pipetted onto it's respective plate. Oluchi Chigbu 10/3/17 10/5/17 We had to repeat the same steps from last because we didn't find phage. The filtrated bacillus phage with the bacteria was taken to conduct a series dilution to 10^-2. Each small tube from 10^-1 to 10^-2 contained 90ml of phage buffer and while conducting the series dilution 10ml is transferred. Each dilution has it's own plate with a total of 6 plates. On each plate a mixture of bacillus bacteria and TSP top agar were poured on the plate. Then 10ml of each dilution was pipette onto it's respective plate Oluchi Chigbu The bacillus purification found a phage however it was found to be contaminated. The small tube containing the picked phage plaque from last time was added to a filtrate with 100ml of page buffer and into a new small tube. Then with the new filtered phage plaque, a series dilution was conducted from 10^0-10^-5. Each dilution was spot tested onto it's own plate.

5 Ayshah Asmat 10/12/ B-DAY PHAGE DAY Today 5 mil of phage buffer was poured over the petri dish found with phage and contaminats (found on 10/5/17) with a pipette. The phage buffer was left atop for 15 min. Then with a pipette without touching the actual phage, the phage buffer was removed and pipetted into a filter. The filter was then transferred into a small tube. With our new filtered solution, a series dilution was conducted to 10^-5. Each series dilution was added to its own bacillus bacteria respectiveley. Then each each tube containing its own dilution and bacillus bacteria had top agar poured into it. That was then poured onto each petri dish for a total of 6 petri dishes. Ayshah Asmat 10/12/ Results: We had plaques from filtered solution obtained last class from a contaminated plate. My 10^0 and 10^-1 plates had plaques on them that I could see when I held my plate up to the light. They look a little more clearn than the lawn background. When the plate is sittingon the bench, the plaques look dark because of the black benchtop. They are about 1mm in diameter and cloudy. My 10^-3 plate was contaminated. The 10^-4 and 10^-5 lawns were smooth and without plaques. A plaque was picked from last classes purification from the plate 10^-2. The plaque was placed in a small a tube with 100ml of phage buffer. A serial dilution from 10^0 to 10^-5 was conducted with the small tubes. TBS bacillus top agar was added to bacillus bacteria and poured over the plate. A spot test was conducted from the serial dilution done. Next class: If I find nice phages, I will conduct a lawn of 6 plates with the small tube containing the picked plaque.

6 AJ We picked a plaque from the plate 10^-2 bacillus. The picked plaque was then set in 100ml of phage buffer. A series dilution was conducted with the small tube of picked plaque and phage buffer. The series dilution was only done up to 10^-2. Each series dilution was dropped in the bacillus bacteria. Specifically 100ml of each dilution into each's respective tube. The tube was then filled halfway through with top agar. The mixture was poured onto the petri dishes. This is the final attempt to find bacillus phage. We find phage but it's usually infested with a lot of bacteria. I believe the bacillus phage is the type to be helping the bacteria produce rather than produce itself. Ayshah Asmat 10/26/17 Picture of purifications done so far.

7 Today she give us a new Lysate named Cove. First the pipette was dipped into the phage and back into the small tube containing 100mL of lysate. We waited five minutes after that added 90 ml of phage buffer to every small tube labeled from 10^-1 to 10^-5. While pipetting it was made sure a new tip was exchanged for every dilution. Then a series dilution was done from 10^0 to 10^-5. Top agar mixed with BTK and the dilutions was added to the petri dish and we waited till the top agar set. Then we pipetted each series dilution from 10^0 to 10^-5 onto the petri dish. This was all done under flame for contamination purposes. Ayshah Asmat /7/17 The petri dishes from 10^0 to 10^-5 all looked similar in the bacteria and phages couldn't be told apart. The cove 10^0 was then diluted again today to 10^-5. The series dilution this time was spot tested rather than tested on it's own plate. A lawn of BTK and top agar was poured onto the petri dish. Then the spot tests were done, and rested.

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9 Ayshah Asmat 11/7/17 11/9/17 The cove lysate went through a dilution only up to 10^-2. 10ml of of 10^-1 dilution were placed in 3 individual tubes containing BTK bacteria, and 10ml of 10^-2 were placed in 3 individual tubes containing BTK bacteria. The tubes were left to set for 15 min. Top Agar was then poured in each tube, and each lawn was poured on to the petri dish. They will incubate until next class. Rahul Warrier 11/9/17 11/14/17 Pre-Lab: We have webbed plates! The webbed plate are flooded and 10ml of the filter-sterilized phage lysate was transferred into a centrifuge tube. Let plate incubate 37C for 30 min. 4.0 ml of phage precipitant solution is added to the nuclease-treated lysate, and then spun at 10,000xg for 20 min. Drain excess liquid from pellet. On Monday, I will add nuclease and phage precipitate solution to all of the lysates, and leave them overnight at 4 degrees C (refrigerator) On Tuesday before class, I will spin these tubes to collect a phage pellet. You will be doing a DNA prep from the phage pellet in class. Lab-Day: DNA purification; 0.5mL of sterile ddh2o is added to the pellet. 2mL of pre-warmed PCR DNA Resin. The pellet is suspended and "dissolved" by taking a pipette and pipetting up and down until everything become a somewhat dissolved mixture. Make sure no bubbles occur. Swirl to mix. That solution is now taken through a filter leaving only it's DNA behind isolating the phage genomic DNA ml of water-resin-phage-genomic DNA is added to each column. We came out to have two columns. The columns were spun in a micro-centrifuge at 13,000rpm for a minute. Remove the liquid

10 from the tube and you're left with DNA inside the column. Repeat with remaining lysate. Add 0.5mL of isopropanol (alcohol) to the column. Spin at 13,000rpm again for a minute.

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12 Oluchi Chigbu 11/14/17 11/16/17 We went upstairs to record phage lysate nubers up in the lab. My Data: Sample ID User ID Date Time ng/ul A260 A / /230 Constant Cursor Pos. Curso ZA50 Default 11/16/2017 9:40 AM ZA30 Default 11/16/2017 9:41 AM ZA30 Default 11/16/2017 9:43 AM ZA30-2 Default 11/16/2017 9:44 AM /28/17 do you have enough dna to do a restriction digest? -Yes Today, we used ZA 50 DNA 1 ( count) in order to perform a restriction digest with the enzyme EcoR1 and no enzyme. We had to figure out how much DNA needed. We did the calculation 500/ =0.72 microliter of DNA was needed. For water we did the calculation = 15.4 microliter of water needed. After that we pipetted 0.72 micro-liters of DNA and 15.4 micro-liters of water into a micro-centrifuge tube named 1 together for the restriction digest. Then, we got buffer for EcoR1 and pipetted 2 micro-liters of it into 1 micro-centrifuge tube. At the end we retrieved a 10x BSA and pipetted 2 micro-liters of it into the 1 micro-centrifuge tube. We did the same steps for EcoR1 we named this tube 2 and at the end added 0.5 microliters of EcoRI into the tube. For HAE3 we do the same steps again but at end we add 0.5 microliters of HAE3 into the tube named 3.

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14 Oluchi Chigbu /30/17 Today 0.3ml of blue gel was placed in our DNA tubes. We took our blue gel DNA and pipetted 10ml into 1% agarose gel. We then took our lysate and evenly split it in the archived tubes. The archives were then labeled with our phage name of 2017VACVTuttiFruityZA. Oluchi Chigbu 11/30/17

15 12/05/17 Went over Journal Club 3, found lysate spot test and old titer. 12/07/17 PHAGE CUPCAKES! -Presentations