Rationale: After my first round of purification I need to calculate the titer of my lysate. However, since not enough TA is available to make multiple plaque assays I conducted a spot test to calculate my titer.

Procedure:

1. Flooded plaque assay from Wednesday for 2 hours by pouring 8mL of phage buffer on the plate then putting it on a shaker.
2. Filtered the phage buffer with a .22µm syringe filter.
3. Diluted with phage buffer out to a dilution factor of 10^-8.
4. Marked a plate to spot for dilutions of 10^0 to 10^-7.
5. Made top agar with 2mL of LB broth, 2.5mL TA, 22.5µL of CaCl2, and 0.5mL of arthrobacter.
6. Let the plate sit until solidified.
7. Spotted each dilution in marked location with 4µL of corresponding lysate.
8. Let spot dry then inverted and placed into incubator.

Observations:

The section with a dilution factor of 10^-7 had 16 plaques. From that I calculated that my lysate has a titer of 4e10. Since  the plaques had a radius of 0.75mm it will take 8µL of 10^-5 lysate to web  a plate.

Conclusions and Next Steps: Since I have a high titer I now have to make a couple webbed plates so I can collect enough lysate to run DNA extraction and then archiving.

Rationale: I now want to purify my phage so I will pick a plaque and run a plaque assay on it to try to get a single type of phage.

Procedure:

1. Marked a single plaque in my spot test.
2. Obtained a microcentrifuge tube and placed 100µL of phage buffer inside.
3. Stuck a micro-pipette tip into marked plaque and swirled around. a small bit trying to avoid arthrobacter.
4. Stuck tip into phage buffer and swirled around to dislodge phage particles.
5. Diluted twice by taking 10µL of next most dilute sample and placing it into 90µL of phage buffer.
6. Enriched each dilution for 30 minutes in 0.5mL of arthrobacter.
7. Made top agar by mixing 8mL of LB Broth, 90µL of CaCl2, and 10mL of 2XTA then plated 4.5mL as a control.
8. Put 4.5mL of top agar into each tube of arthro bacter then plated 10^0 then 10^-2 dilution.
9. Accidentally plated 10^-1 on top of 10^-2 dilution.
10. Inverted both the other plates and placed incubator for 48 hours.
11. Kept 10^-2 dilution upright and placed in incubator for 48 hours.

Obsercations: I will update observations when I go to open lab this Friday.

Conclusions and Next Steps: Next I need to try to web a plate and obtain a high titer. Once I web a plate I can flood it and get a lot of high titer lysate which I can use to do DNA sequencing and other important procedures.

Rationale: Due to the aggressiveness of my phage I will have to dilute my lysate all the way to 10^-6. Also since there is not much arthrobacter available I will have to conduct a spot. test of each of the dilutions rather than a plaque assay for each.

Procedure:

1. Took 10µL of lysate and placed in a microcentrifuge tube labeled 10^0. Added 90µL of phage buffer.
2. Took 10µL of 10^0 sample and placed into tube labaled 10^-1 and added 90µL of phage buffer.
3. Repeated up until 10^-6.
4. Made top agar by mixing 8mL of LB Broth, 90µL of CaCl2, and 10mL of 2X TA.
5. Divided plate into 6 sections.
6. Added 4.5mL of solution to control plate.
7. Added 4.5mL of top agar to 0.5mL of arthro then plated.
8. Let plate sit for 15 minutes then. spotted dilutions 10^-1 to 10^-6.
9. Placed in incubator for 24 hours. Note: accidentally didn’t leave plate inverted.

Observations:

The spot’s were large and weren’t good plaque’s to pick from until 10^-6. The calculated titer according to the spot test is 2.1*10^9. Also a lot of spots had arthrobacter start to regrow in the center of the plaques.

Conclusions and Next Steps: My unpurified lysate has a high titer. I need to purify from here so I picked a plaque and I’m going to run a plaque assay with it and then go directly to trying to web a plate. I may also try to web a plate with the 10^-6 dilution.

Plaque Assay

11/12/18

Rationale: My plate from the previous plaque assay came back with contaminant, and PCR doesn’t seem to be able to detect even known positives. Therefore, I will be performing another plaque assay in hopes of finding the phage from the spot test last week.

Procedure:

1. Enriched 10µL of McClane soil lysate and Lauren’s Bookstore soil in 0.5mL each of arthrobacter for 30 minutes. Also used 10µL of phage buffer and 0.5mL of arthrobacter for a arthrobacter control.
2. Made top agar by mixing 8mL of LB Broth, 90µL of CaCl2, and 10mL of 2X TA.
3. Put 4.5mL of top agar into control plate then pipetted 4.5mL into each tube of arthrobacter.
4. Plated each tube of solution and let sit for 15 minutes then inverted and placed in incubator for 24 hours.

Observations:

My plate was fully lysed. The arthrobacter lawn looked fine and the TA control was also uncontaminated. Lauren’s sample didn’t have any plaques.

Conclusions and Next Steps: My phage seems to be extremely aggressive so I will have to dilute quit far. I will have to conduct a spot test or multiple plaque assays for each dilution in hopes of finding a decent plaque or webbed plate to either flood or purify from. Also I believe that the increased enriching time definitely had some effect on the ability for me to perceive the presence of my phage in my sample.

Rationale: Due to my lack of time to both purify and amplify my phage I will conduct three plaque assays this class with 20µL, 40µL, and 80µL in order to try to get a high titer lysate sooner.

Procedure:

1. Enriched three vials of 0.5mL of arthrobacter with 20µL, 40µL, and 80µL of lysate and labeled 3 plates with corresponding values.
2. Waited for 15 minutes then begun making top agar.
3. Mixed 8mL of LB broth, 90µL of CaCl2, and 10mL of 2xTA in a 50mL tube.
4. Mixed by pipetting up and down.
5. Poured 4.5mL of top agar into control plate.
6. Aliquot 4.5mL of top agar in to each tube of arthrobacter and lysate and mixed by pipetting up and down.
7. Poured each tube into corresponding plate then swirled around to cover entire plate.
8. Waited for 15 minutes before inverting then placed into incubator.

Observations:

All plaque assays turned up negative.

Conclusion and Next Steps: I will try to run PCR on this sample, and if that also turns up negative I will continue to work on Katherine’s lysate to try to get a high titer.

Multiple Person Spot Test

11/5/18

Rationale: Since we’re waiting on Katherine to calculate the titer of her lysate we decided to run a quick spot test on our remaining soil samples.

Procedure:

1. Marked a plate in four sections with the  initials of each member running a plaque assay and one control section for phage buffer.
2. Top Agar was made for two plates by adding 4mL of LB Broth, 45µL of CaCl2, and 5mL of 2X TA.
3. Poured control plate with 4.5mL of solution
4. Added 0.5mL of arthrobacter to remaining solution and mixed by pipetting up and down.
5. Poured test plate and let sit for 15 minutes.
6. Added 10µL of each test lysate onto allocated areas and 10µL of phage buffer to control area.
7. Let sit for 10 minutes then placed in to incubator without inverting because agar did not adhere to plate.

Observations:

My section had a rather large plaque. Note that the control spot is in the area labeled EB and Emily’s spot is in the area labeled PB.

Conclusions and Next Steps:
It’s a relief finally getting a plaque, but also stressful since I don’t have much time to purify and amplify. Therefore, I’m going to conduct 3 plaque assays with 20, 40, and 80 µL of lysate in hopes of speeding up the process. The plaque is rather large for only a 10µL spot, this could mean that it is already of a high titer. If that is the case I could save a lot of time during amplification.

Rationale: In order to guarantee a webbed plate so we can conduct Transmission Electron Microscopy I have received 1mL of Katherine’s lysate and I will calculate the titer and the amount needed to web a plate. I will place more than the calculated required amount so it will definitely be webbed.

Procedure:

1. Counted plaques on plate and took average diameter of plaque.
2. After calculating titer realized that there wasn’t enough lysate to create a webbed plate so plate was flooded.
3. Added 8mL of phage buffer to plate then parafilmed it and stored at 4ºC overnight.

Observations: 514 plaques were formed with 25μL of lysate. This means that the titer is 2.056 * 10^4. The diameter of the plate was 86mm and the average diameter of a plaque was 0.82mm. This means that the amount of pfu needed to web a plate is 1.1*10^4. So the amount of lysate needed to web the plate is 535μL of lysate.

Conclusions and Next Steps: Now I must make a plate with 1070μL of lysate in order to guarantee a webbed plate.

Rationale: After another negative sample it’s back to the drawing board with another sample. I plan to conduct a multi-well enrichment with the next couple of samples and some of my older samples.

Procedure:

1. Filled a 15mL tube with soil up to the 2mL mark then filled with LB broth up to the 12mL mark.
2. Shook with a vortexer for 15 minutes.
3. Spun at 3000g for 5 minutes then filtered with .22μm filter into a 50mL tube.
4. Added 0.5mL of arthrobacter to 50mL tube and placed into shaking incubator.
5. Collected soil metadata for Soil E:
   1. Placed soil in a 50mL conical vial up to the 10mL mark then filled up to the 30mL mark with DI water and added 3 drops of soil dispersion fluid.
   2. Shook vial while holding gloved hand over top for ~1 minute then let sit for 48 hours.
   3. Massed a weigh boat then placed about 3 grams of soil inside. Let sit for 48 hours.
   4. Placed a small amount of soil into vial and added DI water then shook vigorously for 30 seconds then let sit for 2 minutes.
   5. Placed pH paper inside then removed and compared against pH chart.

Observations: The pH of soil E was 6.0. The percent water of the sample was 21.1%. The soil was 6.67% sand, 83.33% silt, and 10% clay.

Conclusions and Next Step: I will have to collect some more soil and then run a multi-well enrichment in the coming lab periods. I will also have to work on putting all the metadata I’ve collected on to the Google Slides spreadsheet.

Rationale: Now that I have replicated phage DNA, if my sample had phage, running a gel electrophoresis will let me see a band showing whether or not there is phage present.

Procedure:

1. Start by adding 35mL of 1X TBE buffer with 0.7g of agarose powder.
2. Heat until it starts boiling and swirl to mix. Repeat a couple times to fully mix agarose.
3. Let mixture cool to about 55ºC then add 1.7μL of Ethidium Bromide and mix.
4. Pour gel into tray with dams on each side and well comb.
5. Repeat to make control gel.
6. Once Solidified adding a bit of TBE buffer to the top to make it easier to remove comb without tearing gel.
7. Added gel to electrophoresis apparatus and flooded with TBE buffer to fill line.
8. Ran gel at 100V for 40 minutes.
9. Imaged using Bio-rad imaging machine.

Observations: On the left is the gel with our samples on it. On the right is the controls. To the left of the ladder are negative controls and to the right are positive controls. As shown none of the samples had phage present.

Interpretations and Next Steps: Yet another soil sample shown to have nothing. This technique also seems to not be working out as it is possible that the sample contained phages of the clusters in primer mix 3 and the band was too faint to see. Next time I’ll bring multiple samples and attempt a multi-well enrichment.

PCR

Date: 10/22/18

Rationale: Since plaque assays have been consistently turning up negative, I am trying a different methodology to find phage.

Procedure:

1. Spun down lysate at 3000g for 5 minutes.
2. Placed 1mL of lysate into micro-centrifuge tube.
3. Boiled sample to release phage DNA from capsid.
4. Made three PCR mix tubes and labeled each with a circle and a number correlating to each primer mix.
5. Each tube was filled with 12.5μL of Taq polymerase and dNTPs. Added 4μL of primer mix 1 to tube 1, primer mix 2 to tube 2, and primer mix 3 to tube 3. Added 1μL of lysate and 1μL of partners lysate. Filled up rest of tube with 6.5μL of DI water so that volume in tube is 25μL.
6. Labeled another three tubes with 12.5μL of Taq polymerase and dNTPs with a plus and a number correlating to each primer mix for positive controls.
7. Filled each tube with 4μL of primer mix corresponding to its number. Added 1μL of positive control lysate. Filled each tube up with 7.5μL to reach 25μL.
8. Gave the tubes to the TA to be run in the thermocycler.

Observations: When 1μL of lysate was added it didn’t mix with the rest of the solution and just stayed at the top of the tube. This is also the first time we are trying the clear Taq polymerase solution.

Conclusions and Next Steps: The amount of lysate could most definitely be increased. Usually the Taq polymerase used comes from a stock that has the dye already added. This could lead to no DNA being replicated by the Taq, as it might not work. Next class I will conduct a gel electrophoresis to see if I did in fact find phage DNA in my sample.