Hanna Kwon. 8/29/27 Make Enrichment Culture. 8/31/17 Filter Enrichment Culture. 9/5/17 Purify Phages That We Found. phage pic

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1 Hanna Kwon phage pic 8/29/27 Make Enrichment Culture Fill 25 ml of media into 50 ml tube Add.5 ml of bacteria (microbacterium foliorum) Fill the tube with soil sample (up to 35 ml) Grow in 30C in shaking incubator Next class I will filter it and do a spot test. AJ /31/17 Filter Enrichment Culture Add 4 ml of enrichment culture into microcentrifugetube Filter out bacteria (filter is.22 um) Take filtered enrichment and drop.5 ml onto plate with 250 ul of host bacteria Plate number: 2 & 5 Next class if I find a phage I will purify it. If not, I will bring in another soil sample and try again. 9/5/17 Purify Phages That We Found 7. Get 5 microcentrifugetubes and label them from 10^1~10^5 Add 90 ml of phage buffer into the microcentrifugetubes Take filtered enrichment culture and add 10 ml into 10^1 tube Then take 10 ml from 10^1 and add to 10^2 tube (repeat all tubes) Add 5 ml from microcentrifugetube and add to bacteria (wait for 5 mins) Pour microbacteriam foliorum and pour into tube with bacteria Then pour each tube onto plates (6)

2 Next class we will do phage amplification. 9/7/17 Spot Test The lawns from last class were no good. Got 5 microcentrifuge tubes and labeled them from 10^1~10^ Got phage buff and put 90 ml into each microcentrifuge tube Used filtered enrichment culture and put 10ml into 10^1 tube then from 10ml from the 10^1 to 10^2 tube (when done wait 5 minutes) Get plate for spot test (pour microbacteriam foliorum and wait a couple minutes) & (draw lines and number them from1 ~6) Take 5ml each from the microcentrifuge tubes and drop on plate Next class Check if plaques formed Kethan Mahavadi signed off 9/12/17 Make Enrichment Culture Spot test plates got contaminated... Next Class Get new tube (50mL) Fill 25mL of media.5ml of bacteria (bacillus thuringiensis 350) Fill soil up to 35mL Grow in shaking incubator Filter and spot test. 9/14/17 Enrichment Culture Filtering Next Class Took 2ml of enrichment culture Moved to microcentrifuge tube with filter Enrichment Get a smaller tube and fill TSB up to 7mL Take Bacillus Thuringiensis 350 (200nm) Fill soil (up to 15) Grow in shaking incubator Filter the enrichment and if I find a phage purify it 9/19/17

3 Did not find a phage... Making Lawn Take Bacillus(.5mL) and mix with Top Agar(TSB) Pour to the plate and let it sit Spot Test Take the filtered enrichments and drop(5ul) them on the lawn and let sit until Tuesday Filtering Faaica and Ayshamughal filtered our enrichments Next Week Hopefully get a phage and purify it Kaivalya Dandamudi 9/26/17 Enrichment Fill tube(15ml) with media for bacillus (9mL) add bacteira (.5mL) add soil (15mL) made one for Faaica and Ayshamughal Spot test (Faaica and Ayshamughal) Make lawn (bacillus.5ml with Top Agar-TSB) drop (5ul) on lawn 9/28/17 Make lawn make lawn (take.5ml of bacillus and mix with Top Agar-TSB) Filter (Faaica and Ayshamughal) Move 4mL enrichment into microcentrifuge filter out bacteria with filter from filtered enrichment take.5ml and spot test Spot test

4 take.5ul of filtered enrichment and drop on plate 10/3/17 Found phage in Hobby's culture. Need to filter more enrichment. Filter Serial Dilution Get 6 plates and 6 microcentrifuges(lable 10^0~10^-5) Add phage buffer (90ul) to each tube Take 10ul from 10^0 and put in 10^-1 (repeat to 10^-5) Take microcentrifuge tubes from 10^0~10^-5 and put into bacillus bacteria and let sit Put top agar(tsb) into the bacteria tubes Pour on plate Kaivalya Dandamudi 10/5/17 Phage Picking

5 Plaque size: 2~3mm, and turbid. Pick a plaque from 10^-2 Mix with phage buffer in the 10^0 tube Take 10ul from 10^0 tube and put in 10^-1 (repeat to 10^5) Take 10ul from microcentrifuge tubes from 10^0~10^-5 and put into bacillus bacteria and let sit (15mins) Pour Top Agar (TSB) into the bacteria tube Pour tube on 6 plates Next time: If my purification works (has the same size/carlity) then I will move on to amplification. AJ /10/17 Purification pick plaque from 10^-2 mix with plaque buffer in the 10^0 tube take 10ul from 10^0 tube and put in 10^-1 Take 10ul from microcentrifuge tubes from 10^0~10^-5 and put into bacillus bacteria and let sit (10mins) Pour Top Agar (TSB) into the bacteria tube Pour tube on 6 plates Next class: If purification works, amplification Kaivalya Dandamudi 10/12/17 Set up plaque amplifcation and try 50 ul phage infection Amplification

6 Get one microcentrifuge tube and put 10ul of phage buffer in Pick a plaque with the tip of pipet tip and dip it into microcentrifuge tube Take 10ul from microcentrifuge tube and put in bacillus tube (leave weekend) Take 50ul from microcentrifuge tube and put in different bacillus tube (wait 30 mins) Pour TSB into the bacillus tube Pour bacillus tube onto plate 10/17/17 Plating of plaque amplification Plate is clear: did not put into incubator/using bacillus tube from weeked add 500ul of phage buffer to phaque amplification/bacillus culture from Filter the amplification into microcentrifuge tube, this becomes 10^0 sample hopefully with amplified phage Take TSB and pour into bacillus tube Pour TSB onto plate and let it sit to create lawn Take 5ul from microcentrifuge tube and drop onto plate Next class: if we have phage, we hope it's amplified to be able to make a web plate AJ 10/26/17 Webbed plate Take 40ul of phage buffer and put into microcentrifuge tube From 10^0 take 10ul and put into 10^1 (do same with the rest) Take 10ul from each tube and mix with bacillus Wait for 20 mins Add TSB into the bacillus tubes Pour solutions onto plates (3)

7 10/31/17 Get new phage found last year from Kethan (ours is not working) Next Class Make a webbed plate with the new phage Kethan Mahavadi signed off 11/3/17 Serial Dilution NF signed off Add 90ul of phage buffer into microcentrifuge tubes (5) (for 10^0 use the phage) Take 20ul from 10^0 and add to 10^-1 (repeat) Take the phage buffer and add 10ul into the bacillus and let sit Add TSB (Top Agar) into bacillus tube Pour onto the plates 11/7/17 Flooding& Serial Dilution (Krishna) Phage: 1cm long; have tails, clear plaque,.5 wide, some have hallo some don't, faded edges Take 5ml of phage buffer and flood 10^0 Wait for an hour Filter Take 8 microcentrifuge tubes and label 10^0~10^-8 Add 90ul of phage buffer to the microcentrifuge tubes Serial Dilution up to 10^-8 Get a plate and divide to 8 spots for spot test Make lawn for plate (Pour TSB into bacillus and pour onto plate) 11/9/17 Webbed Plate

8 Calculating titer (7 pfu/ 5ul) x (10^3 ul/ ml) x (10^7) = 4x10^10 pfu/ml Making more webbed plates Get 3 plates ready Add 10ul to three bacillus tubes Let sit for 7 mins Add TSB into the tubes Pour onto plates Next class we are flooding the plates NF signed off 11/14/16 Flooding/ Spot Test Results: We got three webbed plates from 10^0 (thursday) Take 5mL of phage buffer and add to the three webbed plates Wait for an hour Make a lawn for the spot test plate Collect solution from flooded plates and filter Serial dilution (10ul each) Spot test

9 Dr. J will do nuclease treatment and phage precipitation Next Class: estimate titer of new lysate Purifiy DNA AJ /16/ ul of ddh2o and add to the pellet Wait for 5 mins Get syringe and add the solution and push out inot waste basket Add 2mL of Isoproponal into syringe and push out into waste basket Get a centrifugetube and add colum Spin (throw out inside solution spin throw out whole thing) Add 50ul of wateinto new mircocentrifugetube (lable 1) spin Repeat #7 but add 30ul instead (lable 2) spin Next Class Get copper piece and place on plate Put 10ul of phage buffer filter 10ul of water filter 10ul of water filter 10ul of radioactivity liquid filter Place back in copper storage C10 Dr. Johnson sequences and show me my concentration 11/21/17 Result oncentrations.txt Concentration of purified phage DNA: Tube 1: 2820 Tube 2: (using this one) Give 500 ng of DNA to Dr. Johnson to run on agarose gel 500/ =.85ul DNA 3ul Dye 115ul water All adds up to 20ul Next class: Restriction digest of phage DNA

10 AJ /28/17 DNA prep gel Enzyme ul EcoR1.5 ul Hae III DNA-500ng.85 ul.85 ul.85 ul 10x buffer 2 ul 2 ul 2 ul 10x BSA 2 ul 2 ul 2 ul Water 115 ul 165 ul 165 ul Restriction Digest Next class Gel NF signed off Going from botton to top (chart) add to microcentrifugetube Restriction enzyme will cut the DNA at specific point 11/30/17 Digest Gel Phage Name: Krishmas Add 3ul of die to the tubes of DNA from last class Let sit in heat block for 2 mins Spin for 20 seconds Add to gel (first row 3-5) Get lysate and add 200 ul to gel electrophoresis Next class Journal Club

11 Kethan Mahavadi signed Phage name Soil Sample Collection GPS:37, , Physical Description: soil is very clay like with some grass blades and compost Air Temperature: 27.8 degrees C Collection Date: 8/29/16 Discovery method Enrichment plating choose: Direct or Enrichment plating Identification of plaque Date: Appearance: Mostly isolated plaque Purification of plaque (Number and method used) Describe final plaque morphology (Clear/turbid/halo/comet/size/multiple, etc.) Conditions for production of web plate Collection of high-titer lysate Titer: Date: DNA purification, how many columns did you use? DNA quantification, Concentration/volume: Concentration: Estimated volume collected: How is your tube labeled? Restriction digest, which enzymes cut? EM Visualization Head Diameter: Tail Length: Submitted Archive Report to bacillus.phagesdb.or g or Insert link here! phagesdb.org (Microbacterium phages) Submitted lysate archive to Dr. Johnson Archive How is your tube labeled?