Rationale: Two plates flooded and the flooded lysate made was used to perform serial dilutions to calculate titer.

Procedure: Plates containing 30µL lysate and 10µL lysate, which were flooded 11/13, were filtered under a vacuum hood. Filtered lysate placed in a 15ml vial labeled Flooded lysate x7 original lysate. 10µL lysate transferred to microcentrifuge cap with 90µL PB. The solution was diluted down to 10^-4. Plaques assays were then created to help count titer. The following recipe was used for the experiment (three plates for experiment and one for the control):

• 2mL LB broth (x4)
• 2.5mL 2X TA (x4)
• 22.5µL CaCl2 (x4)
• Lysate
  • 10µL 10^-2 lysate
  • 10µL 10^-3 lysate
  • 10µL 10^-4 lysate

In a test tube, o.5mL Arthrobacter phage was added with lysate from the above experiment (three total lysates), and 4.5mL of the solution made from the above recipe were added to the test tube. Test tube solution poured onto a plate, which sat for 15 minutes, and then placed in the incubator at 27 degrees Celsius for 24 hours.

Observations: Control from 11/12 experiment was contaminated. Switched LB broth solutions and 2X TA solutions for new ones. New solutions were used for this experiment.

Conclusions: Titer will be calculated if the plate yields countable plaques. If not, amplification will continue via revisiting lysate x7. Amplification will continue until high titer is obtained.

Rationale: Calculate the amount of lysate needed to completely web a plate based on the experiments performed on 11/5.

Procedure: Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. Calculated titer with the equation below:

Determined the titer of the experiment performed, so calculations for how much lysate was needed to web a plate shown below:

In a 50mL vial, 2xTA solution was made for the experiment using the equation below:

• 2mL LB Booth (x4)
• 22.5 microliters of Calcium Chloride (x4)
• 2.5mL 2X TA (x4)
• 500 microliters of Arthro (x3)
• 10^-2  lysate
  • 7.09µL plate 1
  • 14.18µL plate 2
  • 2.127 of lysate 10^-1 plate 3

4.5mL of 2XTA solution was added into a test tube containing 10^-2 lysate + Arthrobacter phage solution and quickly poured onto a plate. Plates sat for 15 minutes to solidify, and then inverted and placed in the incubator at 27 degrees Celcius.

Observations: Plates from 11/5 experiments did not sit all the way causing plaques to tear and the TA solution did not completely cover the plate. The control from the experiment performed on 11/5 was positive.

Conclusions: Calculate pfu/µL on 11/12 to determine high/medium titer. If the experiment performed 11/7 is a high titer, flood the plate, and if the titer is medium, redo experiment, same as 11/7 experiment.

Rationale: Calculate Justin’s titer by performing a series of serial dilutions. Justin has a medium titer, and a titer that is greater than 10^8 will be needed to extract the phage genome.

Procedure:  Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. Justin’s lysate was used to perform titer dilutions. The lysate was added into a microcentrifuge with 90µl of PB and 10µl of Justin’s lysate. The solution (lysate plus PB) was diluted from 10^0 to 10^-6. Once this was done, a 50mL vial was obtained to make the solution needed for the plaque assay. The formula below was used to make the solution for 7 plates (six for the experiment and one for the TA control).

• 2mL LB Booth (x7)
• 22.5 microliters of Calcium Chloride (x7
• 2.5mL 2X TA (x7)
• 500 microliters of Arthro

Added the 2X TA last to the 50mL vials. shook the vial, and quickly pipetted the solution onto each vial containing the Artho/lysate solution. Sat each plate for about 15mins to the solution solidify. The remaining solution that was left in the 50mL vial was used for the control. All the plates sat to solidify for 15 minutes, inverted, and then placed into an incubator at 27 degrees Celcius.

Observations: Justin has a medium titer and high titer calculations will be done on 11/7 lab experiment. The results from the titer dilution performed 11/5 will help determine how much lysate is needed to obtain a high titer of Justin’s lysate. The experiment performed on 10/31 with Justin’s lysate was contaminated. and the experiment was negative. The experiment performed on 10/31 was done to test the titer of Justin’s lysate, but the experiment performed 11/5 will do the same.

Conclusions: A titer will be calculated once the plaque assays develop from the 11/5 experiment (48 hours). A titer of 10^8 or greater is required or the experiment will have to be done again, and variables will be altered.

Rationale: Help Justin to obtain a high titter by performing a plaque assay, and alternating some of the variables to obtain a webbed plate.

Procedure: Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. The formula below was used to make the solution for 2 plates (one for the control and one for the plaque assay).

• 2mL LB Booth (x2)
• 22.5 microliters of Calcium Chloride (x2)
• 2.5mL 2X TA (x2)
• 500 microliters of Arthro
• 588µl of Justin’s lysate

2X TA solution was added into a 15mL vial, and in a test tube with 0.5µl Arthrobacter, 588µl of lysate was added. The test tube solution sat for 15 minutes, and then 4.5mL of the 2X TA solution was added to the test tube. The test tube solution was then quickly poured onto a plate, and the rest of the 2X TA solution was poured onto a second plate as a control.

Observations: Gel Electrophoresis performed on 10/29 came back negative. Obtained lysate from Justin to help obtain a high titer. Justin added 488µl of lysate, but in the experiment performed today, 588µl of lysate was added.

10/31 Control

10/31 Plaque Assay

Results: On Monday 11/5/18, calculate how much lysate is needed to obtain how much lysate should be added to obtain a high titer. Check Justin’s flooded plate to help to ensure his plates will have a high titer.

Rationale: Perform Gel electrophoresis to test the presence of bacteriophage DNA clusters after performing PCR with three primers.

Procedure: 

 Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. 4omL of TBE and 0.8g of Agarose was added into a 250mL hydro flask. The solution was microwaved for 2 minutes and placed to cool. The solution was then poured onto a plate to solidify. Once the solution solidified, TBE was filled up to the wells, and DNA ladder was placed into the first well. Primers 1, 2, and 3 plus DNA dye solutions were added to the next three wells, next to the DNA ladder. Once the solutions were placed into the wells, the VMR  was plugged in, and it ran for 45 minutes and the results were imaged.

Results from Gel electrophoresis

Observations: 

Placing DNA dye into wells was hard since some of the Primer/DNA dye solutions was not placed into the wells, but diffused into the Gel made. Starting the VMR was hard as well since it was not set correctly/turned on/chords not plugged in correctly.

Conclusions:

Gel electrophoresis was nice to do and this test is faster than running multiple plaque assays/spot tests since no contamination factors can ruin a Gel Electrophoresis. If the experiment comes back to be negative:

• Collect new soil
• Perform PCR experiment and alternate the variables.

If the experiment comes back to be positive, perform spot test and plaque assay, knowing that phage is present in the soil sample.

Rationale: Set PCR to amplify the phage DNA to prepare for DNA GEL. DNA GEL will determine the presence of phage in the obtained soil sample A.

Procedure: Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. A burner was then placed to set up the aseptic zone. The lysate was obtained and 1mL was added to a microcentrifuge cap. This boiled for 10 minutes to break up the protein ca[sid so the phage DNA could be extracted. In three microcentrifuge caps, Primers 1,2, and 3 were added. The formula below shows the solution concentration added to the caps (x3 for 3 microcentrifuge caps):

• 12.5 microns Taq
• 2 microns of phage DNA
• 6.6 microns of DH2O

After the solutions were added, the three caps were placed in a thermocycler for 52.5 minutes.

Observations: Control and the experiment performed on 10/22 were both negative. No plaques were present on the plaque assay performed on 10/22. No plaques were available to pick, and PCR was done to test whether the soil sample is positive or negative.

Negative Control 10/22

Negative experiment 10/22

Conclusions: Lab experiment performed on 10/22 was negative due to contaminations. Possible contaminations reasons:

• LB broth and 2X TA are contaminated
• Opening/Closing of microcentrifuge caps/things not done in an aseptic zone.

The lysate was used to perform PCR in preparation for DNA GEL experiment. DNA GEL will test whether or not the soil sample obtained is positive/negative. If the soil sample is negative, new soil will be needed to be tested via PCR/DNA GEL.

Rationale: In preparation for PCR testing, soil A was washed to determine if phage are present in the soil. Plaque assay was performed to purify possible phage.

Procedure: 

Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. A burner was then placed to set up the aseptic zone. Plate labeled 10/17/18 was picked, added to 90 microns of PB, and then added to 0.5 microns of Arthrobacter (solution sat for about 15 mins). In a 50mL vial, the TA solution was made following the formula below (x2, 1 for the experiment and one for the positive control):

• 2mL LB Booth (x10)
• 22.5 microliters of Calcium Chloride (x10)
• 2.5mL 2X TA (x10)
• 500 microliters of Arthro
• (made in a 50mL vial)

4.5mL of this solution was pippeted into the Arthrobacter + plaque solution, which was then quickly poured onto plates. Plates sat for about 15 minutes and then placed in the incubator at 30 degrees Celsius.

Soil Washing

Soil obtained from the walk-in fridge was poured into a 15mL vial (4mL mark). LB broth added and the solution was shaken for 15 minutes. The solution was centrifuged and then placed into the fridge since no filter was available in the lab.\

Observations: 

Plaques were present from 10/17/18 lab experiment. One of the plates was picked and purified in 10/22/18 experiments. The groups control was negative once again. Plaque Assay 10/17/18

Control 10/17/18

Conclusions: 

Washed soil will be used to run PCR. PCR will be run 10/29/18 since filters will be in the lab. Possible contaminations reasons:

• LB broth and 2X TA are contaminated
• Opening/Closing of microcentrifuge caps/things not done in an aseptic zone.

Positive experiments from plaque assays will be used to calculate titer.

Rationale: Pick plaque from a past successful plaque assay, that has been passaged twice.

Procedure: 

Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. A burner was then placed to set up the aseptic zone. Plate labeled 9/28/18 was picked, added to 90 microns of PB, and then added to 0.5 microns of Arthrobacter (solution sat for about 15 mins). In a 50mL vial, the TA solution was made following the formula below (x3, 2 for the experiment and one for the positive control):

• 2mL LB Booth (x10)
• 22.5 microliters of Calcium Chloride (x10)
• 2.5mL 2X TA (x10)
• 500 microliters of Arthro
• (made in a 50mL vial)

4.5mL of this solution was pippeted into the Arthrobacter + plaque solution, which was then quickly poured onto plates. Plates sat for about 15 minutes and then placed in the incubator at 30 degrees Celsius.

Observations: 

Control came back negative once again, and all the plaque assays performed came back negative. Why? Possible conclusions:

• LB broth and 2X TA are contaminated
• Opening/Closing of microcentrifuge caps/things not done in an aseptic zone.

Control 10/15/17

Conclusions: 

Perform PCR 10/22/18 to see if phage is in fact present. This test will show extract the phage’s DNA using PCR and Gel. PCR will be used to make multiple copies of DNA at a specific region. PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest. Gel electrophoresis will be used to see the DNA from PCR, which will prove if phage is in the soil sample in less than 2 hours, Multiple factors could have affected the outcome of the result, but the two predominant factors are the two mentioned above. For the past three labs, the same 2X TA/LB broth was used, and all results were negative.

Rationale: Picked plaque assays from soil sample A, that have been passed for three rounds, but lost during the second-third round of purification.

Procedure:

Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. A burner was then placed to set up the aseptic zone. Three plaque assays were obtained from the walk-in fridge to pick the plates once more. 90 microns of PB was added to three separate microcentrifuge caps, and picked plaques added to the solution, which was then added to 0.5mL of arthrobacter.  The formula below was used to make the solution for 4 plates (three for the experiment and one positive control).

• 2mL LB Booth (x10)
• 22.5 microliters of Calcium Chloride (x10)
• 2.5mL 2X TA (x10)
• 500 microliters of Arthro
• (made in a 50mL vial)

4.5mL of the solution made in the 50mL vial was added to the Arthrobacter + phage solution, which was then poured onto a plate. Plates sate for 15 minutes and then inverted and placed in an incubator at 30 degrees Celsius.

Control 10/15/18

Observations: Soil sample B came back negative. Control was contaminated as wells as soil sample A and B plates. Plates that had a successful passage of phage were obtained to continue the experiment. The three plates were passaged twice but failed the third time of purification due to many factors including contamination.

Control 9/10/18

10^0 Soil A

Soil A Plaque Assay

Soil B Plaque Assay

Conclusions/Next steps: Passage and calculate titter on Wednesday. After the third round of purification, calculate the titter needed to obtain a high titer. If the results of this experiment come back negative, the next step would to pick the plaque again, and perform another plaque assay.

Rationale: Perform Plaque assays for both Soil A and B after washing soil sample B.

Procedure:

Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. A burner was then placed to set up the aseptic zone.

Spot Test 

A spot test was performed for both Michael’s and Cooper’s Soil sample B. Obtained a 50mL vial and used the formula below as the plates solution. After the solution was made within an aseptic zone, the solution was poured onto a plate, which sat for >15minutes. Then 10 microliters of lysate (Soil B) were added to the solidified solution, along with one spot for the negative control (Phage Buffer 10 microliters). Then the plates were placed in the incubator for 48 hours. The formula below was used to make the solution for 1 plate (one for the spot test, which was divided into three parts).

• 2mL LB Booth (x10)
• 22.5 microliters of Calcium Chloride (x10)
• 2.5mL 2X TA (x10)
• 500 microliters of Arthro

Plaque Assay

A plaque assay was performed for Michael’s two soil samples A and B. Since the phage was lost during the passage of phage, the lysate from the beginning of the lab (9/12/18) was obtained to perform a plaque assay, in hopes to passage phage. The formula below was used to make the solution for 4 plates (two for Michael’s plaque assays, one for Cooper, and one for the control).

• 2mL LB Booth (x10)
• 22.5 microliters of Calcium Chloride (x10)
• 2.5mL 2X TA (x10)
• 500 microliters of Arthro
• (made in a 50mL vial)

This was all done within an aseptic zone. Pippeted 10 microliters of lysate into the 500 microliters of Artho. Pippeted 4.5mL of the 50mL vial solution into each tube of arthro + lysate, then quickly poured the solution onto plates. The plates sat for about 15 minutes, and then they were placed in the incubator for 48 hours.

Soil Metadata results:

% Water: 21%

% Sand: 1.5 mL

% Silt: 2mL

% Clay: .5mL

Observations:

The plaque assay that was performed on Wednesday 10/3/18 was contaminated. This was the last plaque available to pick, and the experiments of the results came back negative. This resulted in two plaque assays that were performed 10/8/18 (Soil A and Soil B). All of the controls in the lab came back negative once again.

Conclusions/Next Steps: 

Perform more plaque assays to purify plaques if plaques are present. The previous experiments results resulted in the start of the new soil sample b. Soil sample A lysate was used to perform another plaque assays since it has had positive results, but the passage of plaques have failed from contaminations or the picking of a bubble. Knowing that soil sample A will have positive results ensured that on Wednesday, 10/9/18, it will be passed.