Zoha Malik. Phage name. Soil Sample Collection GPS: N, W. Physical Description: Small sample of soil from near the coastline

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1 Zoha Malik Research habits of the mind- for knowing how your lab partner should correctly use the syringe! Research habits of the mind- for your phage lab workout during DNA prep Summary Techniques and Data Table: Please copy this table onto your own wiki page. It is your responsibility to notify me or your TA of updates to this table! Phage name Soil Sample Collection GPS: N, W Physical Description: Small sample of soil from near the coastline Air Temperature: 78 degrees Fahrenheit 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 Yes. 2017VACV/TuttiFruity/ZA. How is your tube labeled? This is my favorite phage picture 8/29/17 First get a conical tube and label it with name, date, and sample name. After that fill it with 25mL of media and.5 of microbacterium foliorum, Then fill the conical tube up to 35 ml with the soil sample and shake the tube. We will leave it overnight so it can work its magic. Next class we will spot test and hopefully we find a phage. Oluchi Chigbu 08/31/17 8/31/17 First we got our soil sample and after that we intsert 1mL of solution 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. Next class we will see if there is a spot. If there isnt a spot we will have to redo it. Oluchi Chigbu 8/31/17 9/05/17 Today we found a phage in our filtered soil solution that was placed on a agar. After that we took our filtered solution and we did a serious of filtration from 10^0 to 10^-5. Then we placed 90mL of phage into each microtube. After that we placed 10 ml of the filtered microtube into the test tube of micro bacterium foliorum. After that we had to wait 15 minutes Then we added the agar to the test tubes and we plated. There were a total of 6 plates and we did the same steps for all of them. Oluchi Chigbu 9/05/17 9/7/17 Today we got our plates and found out all of them are contaminated. We will repeat the same steps from Tuesday. We placed 90uL of phage buffer into each micro-tube. A series dilution was done from 10^0 to 10^-5. After that top agar was placed into the test tube of micro bacterium foliorum. After that we placed the agar in the petri dish. We labeled the dish into six sections. We took 10uL of our dilution and finally we conducted a spot test.

3 Oluchi Chigbu 9/7/17 9/12/17 So today we had to start from the beginning. First get a conical tube and label it with name, date, and sample name. After that fill it with 25mL of media and.5 of microbacterium foliorum, Then fill the conical tube up to 35 ml with the soil sample and shake the tube. We will leave it overnight so it can work its magic. Next class we will spot test and hopefully we find a phage. Ayshah Asmat 9/12/17 9/14/17 So today we did filtration. We first removed the plunger from the fringier. After that we attached the barrel of the syringe to the sterile filter. Then we pipetted the 1mL of supernatant from the enriched culture and we transferred the supernatant to the barrel. Once we were done with. We placed the top of the filter into the tube and then released the sterile filter. After that we pipetted 1mL of sterile filtrate. Spotted the sterile filtrate soil sample onto the plate. Ayshah Asmat 9/14/17 9/19/17 The filtration happened first by removing the plunger from the fringier and after injecting the soil sample into the syringe through the pipette. I then transferred 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. The top agar was placed in the petri dish, and we waited for it solidify. Then the filtered sample solution was spot tested. Ouch Chigbu 9/19/17 9/26/17 We found a phage using the bacillus bacteria. First the pipette was dipped into the phage and back into the small tube containing 100mL of phage buffer. 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 we would make 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 pipetted each series dilution from 10^0 to 10^-1 onto the petri dish. This was all done under flame for contamination purposes. Oluchi Chigbu 9/26/17 9/28/17 We had to repeat the same steps from last because we didn't find phage. Which was caused by not adding the bacillus bacteria to the TSB top agar. Oluchi Chigbu 9/28/17 10/3/17 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

4 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 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 10/5/ 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 10/10/17 10/12/17 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. AJ We picked a plaque today from the plate 10^-2 containing phage, and placed the picked plaque into small tube containing 100 ml of phage buffer. The small tube went through a series dilution only up to 10^-2. The dilution were each poured into their own tube containing bacillus bacteria. The tubes then had top agar poured in them. They were then each poured onto their petri dish. This is our final attempt to find a bacillus phage.

6 Ayshah Asmat 10/26/ 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 we would make 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^-5. Top agar 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 11/2/ 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.

7 Ayshah Asmat 11/7/17 11/14/17 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 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.

8 Oluchi Chigbu 11/14/17 11/16/17 Today we went up stairs to the lab and we recorded our phage lysate number. 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 Oluchi Chigbu 1130/17 11/28/17 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.

9 11/30/17 Today we took 0.3 ml of blue dye and added it to our DNA. After that we took 10ml DNA and added that to 1% agarose gel. We also got our lysate and we spilt it half and we put the lysate in the archived tubes. The archives were then named with our phage 2017VACV/TuttiFruity/ZA. Oluchi Chigbu 11/30/17 12/05/17 Today we went over journal club 3.