Rationale: Melissa wanted more lysate from her soil that tested positive for phage so Lily and I washed and enriched more soil

Process:

• Wash table and set up aseptic zone
• Added ~7 mL LB broth to soil (filled to 5 mL mark)
• Shook and vortexed for 15 minutes
• Centrifuged for 10 minutes at 3000 G
• Filtered with syringe filter
• LCG sample enriched and incubated for 48 hours at 28 degrees C
• RSM sample stored as direct in fridge
  • there was not enough arthro for both samples to be enriched

Next steps: Perform plaque assay with enriched sample to check for any plaques

Rationale: Based off results of spot test, decided whether or nor to continue using soil sample 8

Plate 2 for soil sample 8 spot test completed 10/31/18.

Clear control plate for spot test from 10/31/2018.

Plate 1 for soil sample 8 spot test completed 10/31/18.

Conclusion and Next Steps:

the spot tests showed no sign of phage presence, therefore soil sample 8 will be discontinued.

Rationale: perform plaque assay with plaque 1 because the previous plaque assay had a contaminated control plate

Plaques assay 1 for p1.                                                 

Contaminated control for p1 plaque assay 1.

Process:

1. Added 30 µL of p1 lysate into o.5 mL arthro and let sit for 15 minutes
2. Made LB agar media for 4 plaque assay plates (control, 3 samples)

   1. Reagents	Amount
      LB Broth	8 mL
      2X TA	10 mL
      1M CaCl2	90 µL

3. Poured 5 mL of media into 3 separate tube for sample plates
   1. One for RSM, LCG, and SJ
4.  Poured Arthro and lysate mixture into an LB agar sample tube
5. Poured contents of tube onto a plate
6. Poured the contents of the remaining LB agar media without lysate onto control plate
7. Let solidify for ~15 minutes
8. Incubate for 48 hours at 28 degrees C

Next Steps: calculate titer of lysate and perform another plaque assay to get a higher titer.

Rationale: perform plaque assay with plaque 1 to produce first plate with phage

Process:

1. picked a plaque from Melissa’s plate with phage
   1. touched pipette tip to the center of plaque
      1. touched a little bit of the surrounding arthrobacter
   2. swirled tip into tube filled with 70 µL of phage buffer
2. Added 30 µL of phage buffer + phage lysate into o.5 mL arthro and let sit for 15 minutes
3. Made LB agar media for 3 plaque assay plates (control, 2 samples)

   1. Reagents	Amount
      LB Broth	6 mL
      2X TA	7.5 mL
      1M CaCl2	68 µL

4. Poured 5 mL of media into two separate tube for sample plates
   1. Ended up disposing of one of the tubes
5.  Poured Arthro and lysate mixture into a LB agar sample tube
6. Poured contents of tube onto a plate
7. Poured the contents of the remaining LB agar media onto control plate
8. Let solidify for ~15 minutes
9. Incubate for 48 hours at 28 degrees C

Next Steps: calculate titer of lysate and perform another plaque assay to get a higher titer.

Rationale: Complete spot test with new sample isolations and the re-purified products from the heating and killing bacteria method to check for plaques.

Process:

1. Set up aseptic system
2. Made LB agar media for 1 control plate and 2 sample plates

   1. Reagents	Control	Sample
      LB Broth	2 mL	4 mL
      2x TA	2.5 mL	5 mL
      CaCl2	23 µL	45 µL
      arthro	NA	1 mL

3. poured 5 mL of LB agar media into each plate and let sit ~5 min
4. filtered enriched sample with syringe filter
5. pipetted 5 µL of direct, enriched, RSM1, SJ1, LCG1, RSM2, SJ2, LCG2, and phage buffer (negative control) into designated areas of sample plate and let sit ~10 min
   1. the initialed samples are the results from the heating and killing bacteria method
   2. the 1 indicates arthro was added and the 2 indicates arthro was not added (except in the case of LCG2, where arthro was added)
6. incubated for 48 hours

Next steps: If we get plaque we may try to get a high titer lysate  from our own sample, but we will most likely just use a classmate’s lsyate and start to work towards getting higher titer by first performing a plaque assay.

Rationale: Centrifuge and filter contaminated samples and enrich soil to get fresh direct and enriched isolations using filters.

results from the heating and killing bacteria method

The results showed high levels of contamination, but we were advised to centrifuge and re-filter the samples and then plate them

Process: 

Soil Metadata

• mass of weigh boat and dry soil = 8.651 g
• mass of weigh boat and wet sample = 14.225 g
• mass of weigh boat = 2.315 g
• mass of H2O
  • 14.225 g – 8.651 g = 5.574 g
• mass of wet soil
  • 14.225 g – 2.315 g = 11.910 g
• percent water
  • 5.574 / 11.910 * 100% = 46.8% H2O

Re-purification of Lysate

1. transferred ~1 mL of samples from plate wells into micro-centrifuge tubes
   1. wells with arthro were put into one tube and the wells without arthro were put into another
2. the tubes were centrifuged for 2 minutes at 13,300 G
3. supernatant was emptied into a clean dish and then filtered with a syringe filter into two fresh micro-centrifuge tubes
4. filtered lysates were stored in the fridge

Soil Enrichment

1. a 15 mL conical tube was filled with soil to the 3 mL mark
2. LB broth was added to the 12 mL mark
3. mixture was shaken and vortex-ed for ~10 minutes
4. tube was centrifuged for 5 minutes at 3,000 G
5. the supernatant was filtered using a syringe filter into two 15 mL conical tubes
   1. One tube was labeled direct and stored in the fridge
   2. 0.5 mL of arthro was added to the other tube (enriched) and it was left in the shaking incubator at 27 °C and 150 RPM

Observations:

The enriched sample had very little lysate in it – only 1.5 mL.

Next Steps:

Perform a spot test with the new direct and enriched isolations, as well as the re-filtered lysates (arthro and no arthro) from the previous week.

Rationale: Wash new soil samples. The lab was out of filters so we designed a protocol to heat and kill the bacteria.

Process:

1. ~3 mL of soil and ~9 mL LB broth were added to tube and vortexed and shaken for 10 minutes
2. the mixture was centrifuged for 5 minutes at 3000 G

Heating and Killing Bacteria Method

1. A hot plate was heated to 55° C
2. Using a transfer pipette, supernatant from the centrifuged lysate was added to the wells of a plate (3 wells per lysate)
3. The sample plate was heated for 5 minutes
4. The hot plate was turned up to 60° C
5. The sample plate was left to heat for about 15 minutes
6. The sample plate was taken out to cool and then 100 µL of arthro was added to 2 wells for every 3 belonging to 1 lysate
   1. The sample plate MAY not have cooled down enough
7. The hot plate was turned down to 27° C and set to shaking at 150 RPM
   1. The sample plate may also have been returned to the hot plate before the hot plate had been cooled sufficiently
8. The sample plate was to left to incubate until the next class

Soil Metadata

• mass of weigh boat = 2.315 g
• mass of weigh boat and wet soil = 14.225 g
• soil sample was left under hood until next class

Next Steps

plate the enriched isolation to test if the heating and killing bacteria method was successful. In addition, wash soil samples again with filters.

Rationale: run pcr reactions on a gel to see if phage DNA was found and replicated in the soil sample

Process:

1. 0.8 g of agarose were dissolved in 40 mL TBE (Tris/Borate/EDTA) buffer
   1. a microwave was used to heat the mixture in order to dissolve the agarose
2. Once the mixture cooled, the TA added Ethidium Bromide
3. The mixture was poured into a gel tray and left to solidify
4. the solidified gel was then placed in a gel box and submerged in TBE buffer
5. the pcr reactions and ladder were pipetted into the wells according to the following table

   1. lane	sample	amount
      1	RSM & SJ PM1	10 µL
      2	RSM & SJ PM2	10 µL
      3	RSM & SJ PM3	10 µL
      4	Ladder	5 µL
      5	shared	10 µL
      6	shared	10 µL
      7	shared	10 µL

6. the electrodes were connected to the gel box and the gel was run for about 30 minutes
7. Once the yellow marks had moved about 3/4 of way down the gel, the gel was taken out to be photographed with the gel imaging machine

Results:

gel run on 10/22/2018 showing only bands from the ladder

Next Steps:

The results showed no sign of phage DNA in our soil samples so my group decided on collecting a new sample and enriching yet again

Rationale: To conserve resources, we ran a pcr test on our enriched isolations to check for the presence of phage DNA.

Process:

1. Wash table
2. centrifuged enriched isolation
   1. 5 minutes at 3000 G
3. added ~ 1 mL to microcentrifuge tube
4. gave tube to Lathan to boil
5. Prepared 4 different PCR reactions as shown in the table below

   1. Reagents	Tube 1	Tube 2	Tube 3	Pos. Control
      TAQ Polymerase	12.5 µL	12.5 µL	12.5 µL	12.5 µL
      Primer Mix	4 µL PM1	4 µL PM2	4 µL PM3	4 µL PM1
      DNA RSM	1 µL	1 µL	1 µL	NA
      DNA SJ	1 µL	1 µL	1 µL	NA
      Pos. Control DNA	NA	NA	NA	1 µL
      dd H2O	6.5 µL	6.5 µL	6.5 µL	7.5 µL

6. Gave tubes to Lathan to run PCR cycle in PCR machine

Next steps: Run PCR products on a gel electrophoresis to check for bands that could indicate phage DNA. Also, calculate % sand/silt/clay.