Previous Results:

• DNA extraction was recently completed and DNA was obtained from the phage sample

Objective:

• Set up PCR using the concentrated DNA sample and prepare to run a gel test

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. The DNA microcentrifuge tube “Pippa” was obtained and 3 microcentrifuge tubes were labeled 1-3
3. 2 microliters of DNA was added to each tube, along with 12.5 mircoliters 1x Master Mix, and 6.5 microliters ddH2O
4. Each tube, labeled 1-3, was given 4 microliters of primer. Tube 1 had Primer 1, Tube 2 has Primer 2, and Tube 3 had Primer 3. Each tube had a total of 25 microliters liquid
5. The tubes were then placed in a thermo-cycler @ 98.0 degrees Celsius for 5 minutes, then completed 35 cycles of 94 degrees Celsius for 30 seconds, 55.1 degrees Celsius for 30 seconds, and 72 degrees Celsius for 45 seconds. The process was completed with 5 minutes in 72 degrees Celsius
6. The tubes were then placed in the freezer at a temp of 4 degrees Celsius until next lab

Results:

• PCR and gel were not completed, therefore there are no results to report

Next Steps:

• During the next lab a gel will be ran on the DNA and results will be obtained

Previous Results:

• The final result of the phage precipitation was a pellet in the bottom of the 50 mL tube. This was the condensed phage that will be used for DNA extraction

Objective:

• To complete DNA extraction without contamination and obtain enough DNA to continue analyzing it
• Have enough time to complete the nanodrop process and have positive results

Procedure:

1. The 50 mL tube containing the phage pellet was brought to room temp
2. 0.5 ml sterile water was added to tube and mixed to resuspend the pellet
3. 2 ml of DNA Clean-Up Resin at 37 degrees Celsius was added and mixed
4. Pellet was separated equally into 2 microcentrifuge tubes and spun at 12.5 x 1000 g for 3 minutes
5. Supernatant was removed from the microcentrifuge tubes without disturbing the cloudy precipitant (DNA) and add 1 ml 80% Isopropanol to the tubes each
6. Step 5 was repeated 2 times, then once more but without removing the alcohol
7. Solution was then transferred to two columns and the vacuum was used to filter the liquid
8. Columns were centrifuged at 12,000 g for 5 min
9. 100 microliters of Elution Buffer at 80 degrees Celsius was added to each column. Left to sit for 1 minute
10. Columns were centrifuged at 12,000 g for 1 minute
11. Two tubes of DNA sample were combined into 1 microcentrifuge tube and labeled. Phage was named “Pippa”
12. The nanodrop machine was then used by adding a drop of Elution Buffer to the machine to prepare for DNA drop
13. DNA drop was added and the machine provided results

Results:

• The results of the nanodrop showed the sample of DNA was not concentrated enough and had a nucleic acid reading of 43.1 ng/uL

Next Steps:

• A PCR will be dome on the DNA sample and a gel will be completed

Previous Results:

• The spot test (11/14) with dilutions through 10^-8 of Lysate #8 was examined. All dilutions were lysed, therefore the titer could not be calculated. Dilutions of 10^-8 will be have to be made since the titer is a high titer and can not be calculated with a spot test.

Objective:

• Complete the first step of DNA extraction by obtaining a phage precipitate

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. Lysate #8 was obtained and 10 mL was put into a 50 mL tube
3. 40 microliters of nuclease was added to the lysate and tube was inverted 10 times to mix
4. 4 mL of phage precipitate was added to 50 mL
5. Mixture was placed in 37 degrees Celsius for 30 min, then room temp for 40 min
6. The 50 mL tube was then massed (29.57 g) and a 50 mL tube was filled with water of equal mass (29.63 g) to prepare for centrifuge
7. The tube was then centrifuged at 10,000 g for 20 min, in 4 degrees Celsius
8. Precipitate was then placed in -20 degrees Celsius freezer until next lab

Results:

• Experiment was not complete, therefore there are no results to report

Next Lab:

• During next lab, the second part of DNA extraction will be completed. Further dilutions of Lysate #8 will be made to calculate the titer of the lysate and move on to a TEM.

Previous Results:

• On 11/13 the plaque assays from 11/12 were flooded by Dr. Adair. The plates were completely lysed, meaning the lysate may be a high titer

Objective:

• The lysate from the flooded plates will be filtered and a spot test of dilutions will be made to later calculate the titer of Lysate #8

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. Flooded plaque assays from 11/12 were obtained and lysate was filtered using a top filter and vacuum. 25 mL of Lysate #8 was collected from 4 plates
3. Overlay agar for spot test was mixed using 2 mL LB broth, 2.5 mL 2x TA, 0.5 mL Arthrobacter, and 22.5 microliters CaCl2
4. Overlay was plated and left to harden for 15 min. grid was drawn on plate, making 9 spaces
5. Lysate #8 was obtained and dilutions were made out to 10^-8. 90 microliters phage buffer was added to microcentrifuge tube labeled “10^-1” and 10 microliters of lysate was added. Then 10 microliters of “10^-1” was added to tube containing 90 microliters phage buffer, labeled “10^-2.” Process was repeated through 10^-8
6. 10 microliters of each dilution (10^0 – 10^-8) was pipetted onto the hardened plate, in their separate spaces in the grid.
7. The plate and dilutions were left to sit for 10 min. Then incubated for 48 hours

Results:

• The experiment was not completed, therefore there are no results to report

Next Steps:

• During next lab, the spot test will be examined. It will be determined which dilution to make a plaque assay with to calculate the titer. After the titer of the lysate is known, TEM will be conducted.

Previous Results:

• On 11/8 the plates made of “Lysate #6 10^-2” were flooded. On 11/9 the lysate was collected (Lysate #7) and plaque assay was made by a lab partner. Today, the plate was examined, and although the control did not have any contamination, the assay looked contaminated, as if there were no plaques.

Objective:

• Plaque assays of serial dilutions for Lysate #7 will be redone, due to the results of the last assays. Hopefully a titer can be calculated using these plates

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. Lysate #7 was obtained and diluted out to 10^-3
3. Using 90 microliters phage buffer and 10 microliters of lysate in a microcentrifuge tube, the 10^-1 lysate was made. 10 microliters of 10^-1 was transferred into 90 microliters phage buffer to create 10^-2. This was repeated to make 10^-3
4. 10 microliters from each dilution (10^0 – 10^-3) were added to individual tubes of 0.5 mL Arthrobacter. Left to infect for 10 min
5. Overlay Agar was mixed in a 50 mL tube using 10 mL LB broth, 12.5 mL 2x TA, and 112.5 microliters CaCl2
6. 4.5 mL of Overlay was added to each Arthro tube, leaving 4.5 remaining in the 50 mL tube used for control plate
7. All 5 agars were plated, left to harden for 10 min, and incubated for 24 hours

Next Steps:

• During the next lab, the plates will be examined and used to calculate the titer of Lysate #7. Depending on the titer, TEM will be run on the sample

Previous Results:

• During the last lab, while the lysate was take to do TEM, plaque assays were create by the lab group. They made 5 plates using Lysate #6 10^-2. The control plate did not have contamination. 2 plates had slipped and one plate looked as though Arthrobacter was not mixed in the overlay Agar. It was determined that the procedure should be repeated.

Objective:

• Create plaque assays using 10^-2 diluted Lysate #6 without slipping and contamination

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. Lysate #6 was obtained and a 10^-2 dilution was remade. Microcentrifuge tubes labeled “10^-1” and “10^-2” each had 180 microliters phage buffer
3. 20 microliters of Lysate #6 was added to “10^-1” tube, then 20 microliters were taken from that tube and put into the “10^-2” tube
4. After mixing the tube, 20 microliters of 10^-2 lysate was added to 5 tubes containing 0.5 mL of Arthro and left to infect for 10 min
5. An Overlay Agar was mixed using 12 mL LB broth, 15 mL 2x TA, and 135 microliters CaCl2
6. 4.5 mL of Overlay Agar was added to each Arhtro tube, leaving 4.5 mL of Overlay in the tube for a control plate
7. The mixtures were then plated (6 plates: 5 experimental and 1 control) and left to harden for 15 min, then incubated for 48 hours

Results:

• The plaque assays were not completed, therefore there are no results to report
• One of the experimental plates experienced plate slipping, but was incubated with the other plates

Next Steps:

• During the next lab, the plates will be flooded, the lysate collected, and plaque assays created to try and calculate a high titer
• A TEM will be run again on Lysate #6 using another staining agent

Previous Results:

• The plaque assays with the serial dilutions of Lysate #6 were examined. The control was not contaminated and plates 10^-1 and 10^-2 were completely lysed. Plates 10^-3 and 10^-4 had a good number of plaques that can be used in a titer calculation.

Objective:

• To calculate the titer of Lysate #6
• Run TEM on the lysate and obtain a picture of the phage in the sample

Procedure: Titer Calculation

1. Plate “10^-3” had 680 plaques on the plate
   1. (680 pfu/10 microliters) x (10^3 microliters/mL) x (10^3) = 6.8 x 10^7 mL = Medium Titer

Procedure: TEM

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. 0.5 mL of Lysate #6 was obtained and put into a microcentrifuge tube
3. Parafilm was placed on the cover of a plate and 1 drop (20 microliters) of lysate, 2 drops H2O, and 1 drop uranyLess were put on the parafilm in a line
4. EM grid taken from the well labeled A7 was placed shiny side down in lysate for 5 min
5. EM grid placed on each drop of water for 2.5 min
6. EM grid placed on uranyLess drop for 1 min
7. Liquid was wicked away from EM grid using paper, then the grid was placed back into well A7
8. Grid was looked at under TEM, picture taken

Results:

• It was difficult finding any phage using the microscope. After searching the grid it was noticed that there was phage, but only heads, no tails. Clusters of material were found all over the grid.

Picture of phage using TEM

Next Steps:

• During the next lab, a TEM will be run again but using a dye other than uranyLess to try and stain the phage differently. The same procedure will be used. Since the titer of the lysate is still not high enough, plaque assays and plate flooding will continue.

Previous Results:

• The plaque assays of Lysate #5 were examined and all plates (10^0 – 10^-2) were all completely webbed. The 10^0 and 10^-1 plates had almost zero Arthrobacter left on the plate, meaning it was completely covered with plaques.
• The control plate from last lab did not contain any contamination

Objective:

• Collect the phage from the plates using phage buffer
• Use a plate shaker for efficiently collecting the phage

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. Plaque assays plated with Lysate #5 were obtained and 6 mL of phage buffer was added to each plate (10^0 – 10^-2)
3. The plates were placed on an electric plate shaker for 75 min instead of allowing the phage buffer to sit for 48 hours to collect phage
4. Then the lysate on the 3 plates was filtered into a 50 mL tube using a syringe and filter. All 3 lysates from the 3 separate plates went into the same 50 mL tube
5. The lysate was then labeled “Lysate #6”

Results:

• The experiment was not completed, therefore there are no results to report. Results will be available during next lab

Next Steps:

• During the next lab Lysate #6 will be diluted out to 10^-4 in order for plaques to be counted for a high titer. If a high titer is obtained, then the experiment will move on in the ordered procedure.

Previous Results:

• The webbed plate created 10/26, labeled “Lysate #4 Webbed”, was not completely webbed, meaning the volume used to make the web was not high enough
• The plate, “10^0 #4”, that was flooded (10/26) had completed the 48 hour time period with phage buffer, the experiment will be continued using this collected lysate

Objective:

• Correctly filter and obtain the lysate from the flooded plate
• To make plaque assays and obtain at least one webbed plate to calculate the titer of the lysate

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. The lysate on the plate from last lab (10/26) that was flooded, labeled “10^0 #4”, was obtained using a syringe and filter. It was filtered into a 15 mL tube and labeled “Lysate #5”
3. Serial dilutions were made using Lysate #5, 10 microliters were added to a microcentrifuge tube containing 90 microliters phage buffer. This lysate was labeled “10^-1”
4. Step 2 was repeated until “Lysate #5″ was diluted out to 10^-2
5. 10 microliters of each dilution (10^0 – 10^-2) were added to individual tubes containing 0.5 mL Arthrobacter and left to infect for 10 min
6. Overlay Agar was created for all experimental plates (3 plates) and a control plate in a 50 mL tube using 8 mL LB broth, 10 mL 2x TA, and 90 microliters CaCl2
7. 4.5 mL of Overlay Agar was added to each Arthrobacter tube with lysate and mixed. 5 mL Overlay Agar was left in the 50 mL and used for the control plate
8. All 4 agars were plated and left to harden for 15 min, and incubated for 48 hours

Results:

• The experiment was not completed in this lab, therefore there are no results to report.

Next Steps:

• During the next lab the plaque assays will be examined and if webbed, then the plates will be flooded for a future calculation of a high titer

Previous Results:

• The webbed plate created on 10/22 was not completely webbed, therefore the volume used to create it was not high enough
• The Plaque Assay of Lysate #4 was positive, but had too many plaques to count and calculate the titer. It was realized that serial dilutions should have created at the time of plating Lysate #4 to ensure the titer could be calculated

“Lysate #4” Plaque Assay using 10 microliters lysate

“Lysate #2” Webbed plate using 64 microliters lysate

Objective:

• To replate Lysate #4 and dilute it out to 10^-4 to ensure plaques can be counted for titer calculations
• To corrected make plaque assays with contaminations

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. The “Lysate #4” tube was obtained and 10 microliters was added to a microcentrifuge tube containing 90 microliters phage buffer. This lysate was labeled “10^-1”
3. Step 2 was repeated until “Lysate #4 was diluted out to 10^-4
4. 10 microliters of each dilution (10^0 – 10^-4) were added to individual tubes containing 0.5 mL Arthrobacter and left to infect for 10 min
5. Overlay Agar was created for all experimental plates (5 plates) and a control plate in a 50 mL tube using 12 mL LB broth, 15 mL 2x TA, and 135 microliters CaCl2
6. 4.5 mL of Overlay Agar was added to each Arthrobacter tube with lysate and mixed. 5 mL Overlay Agar was left in the 50 mL and used for the control plate
7. All 6 agars were plated and left to harden for 15 min, and incubated for 48 hours

Results:

• Experiment was not completed, therefore there are no results to report

Next Steps:

• During the next lab, the plates will be examined and the plaque assay with less than 100 plaques will be used in a titer calculation. Once the titer is known the volume needed to web a plate using “Lysate #4” will be calculated and a plate will be made using that result.