Purpose: The purpose of this lab was to become familiar with and understand how to use DNA Master, as well as learn how to auto annotate.

Tools/Procedures:

• Tools
  • DNA Master
  • Elesar
  • PhagesDB

1. DNA Master was successfully updated and installed.
2. The Fasta file for Elesar was obtained from PhagesDB and imported to DNA Master. The file was exported and a sequence entry was made.
3. Auto-annotation was performed on Elesar.
4. The auto-annotations were examined and saved.

Results:

• No annotations were started today.
• DNA Master was successfully opened and used.

Conclusions:
DNA Master was successfully installed and the process of learning how to operate the software was begun. The installation and proper set-up took time, and the software had to be reinstalled. After reinstallation and troubleshooting, the program is successfully running.

Future Work:
For future work, the next step will be to learn how to annotate and begin the process.

Rationale: The purpose of this lab is to complete DNA extraction and quantify the DNA with the Nanodrop.

Description of Procedures:

1. The titer of the lysate was determined to be 2.0 x 10^10.
2. 0.5 ml of sterile water was added to the DNA pellet to re-suspend it.
3. 2 ml of 37 C Clean Up Resin was added to the pellet and swirled.
4. Resin pellet was transferred to 2 microcentrifuge tubes and spun for 3 minutes at 12,500 xg.
5. Supernatant pulled off with a bulb pipette and 1 ml of 80% isopropanol was added to each tube. The tube was spun at 12,500 xg for 3 minutes.
6. Step 4 repeated
7. Supernatant pulled off again and 1 ml of 80% isopropanol added to each tube. Liquid transferred to a two column syringe and allowed the vacuum to pull the liquid through the column to separate the DNA.
8. Column added to a clean microcentrifuge tube and spun at 12,000 xg for 5 minutes.
9. Transferred to a clean tube and 80 ul of 80 C elution buffer added to the tube. It was then spun in the centrifuge at 12,000 xg for 1 minute.
10. DNA was quantified using the Nanodrop.

Observations/Results:

• DNA- 808.978 ng/ul
• A260/A280- 2.049
• A260/A230- 0.193

Interpretations:
The procedure was completed. The DNA was extracted and quantified. The A260/A230 value was low, meaning there could be too much guanidinium thiocyanate left in the DNA. The next step would be to perform PCR and run a gel, but there is not enough time left in the lab.

Extracted DNA and Spot Titer Test:

Rationale: The purpose of this lab is to collect a high titer lysate and perform a spot titer test to calculate the titer, start the DNA extraction process, as well as perform TEM with the phage.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit.
2. The webbed plates were flooded with 8 ml of phage buffer and put in the refrigerator overnight.
3. Lysate was collected from the webbed plates and filtered using a 0.22 um top filter.
4. 35 ml was collected and separated into two tubes for two samples.
5. 10 ml of lysate was poured into a separate tube for DNA extraction. 40 ul of nuclease was added and the tube was inverted 10 times. 4 ml of PEG was then added, the tube was inverted once and then put in the incubator at 37 C for 25 minutes.
6. The tube was then allowed to sit at room temperature for 40 minutes.
7. While waiting, electron microscopy was performed. A TEM grid was made with 20 ul of lysate, two 20 ul dots of DI water, and a drop of uranyl acetate. The TEM grid was allowed to sit in the lysate for 5 minutes, and water for 2.5 minutes. It was then allowed to sit in the UA for 1 minute and then dried.
8. The image of the phage showed it to have an average head size of 58 nm and a tail of 160 nm.
9. A spot titer test was performed to calculate the titer of the lysate collected, with dilutions out to 10^-7. A 5 ml plate was made and inverted and stored in the incubator until the next lab. It was labeled LIP 11-28-18 STT (Spot Titer Test)
10. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations/Results:

• Head of phage: 58 nm
• Tail of Phage: 160 nm
• 35 ml of lysate collected
• Bubbles seen on the control plate.

Interpretations/Next Steps:
The procedure was complete for TEM and spot titer test, but the DNA extraction was not complete. The lysate was not spun in the centrifuge due to an error with the machine and will have to be performed in the next lab. The next step will be to finish DNA extraction and possibly run PCR.

Flooded Plates and Image of Phage:

Rationale: The purpose of this lab is to web plates to obtain more of a higher titer lysate.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit.
2. Lysate 1 was diluted out to 10^-3.
3. Top agar was made for 7 plates. 14 ml of LB broth and 157.5 ml of CaCl2 were added to a tube.
4. 10 ul of 10^-3 lysate was added to 6 tubes of 0.5 ml of arthrobacter and was allowed to sit for 10 minutes.
5. 17.5 ml of 2x TA was added to the tube and mixed. 4.5 ml of the solution was added to each tube of arthrobacter and poured onto 6 plates labeled LIP 11-26-18 webbed plate. The rest of the solution was poured onto a plate labeled LIP 11-26-18 Control.
6. The plates were allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
7. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

• Bubbles on the control plate.

Interpretations/Next Steps:
The procedure was complete. The next step will be to flood the plates to collect a higher titer lysate.

Webbed Plates:

Rationale: The purpose of this lab is to flood webbed plates to create more of a high titer lysate.

Description of Procedures: 

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit.
2. 6 ml of phage buffer was added to each of the four webbed plates. The plates were placed on the shaker and allowed to shake for one hour.
3. The lysate was collected from the plates and filtered with a 0.22 um syringe filter into a tube labeled LIP 11-20-18 Lysate 3.
4. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

• Plates not fully webbed.

Interpretations/Next Steps:
The procedure was complete. However, the plates were not webbed. Due to this the titer most likely will not increase. The next step will be to perform a titer test.

Webbed Plates and Control:

Rationale: The purpose of this lab is to create webbed plates to create more lysate at a higher titer.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit.
2. The titer from the spot titer test was determined to be 7.5 x 10^8.
3. Top agar was made for 5 plates. 10 ml LB and 112.5 ul of CaCl2 were added to a tube.
4. 10 ul of the 10^-4 dilution of lysate 1 was added to four tubes of 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
5. 12.5 ml of 2x TA was added to the top agar solution and pipetted up and down to mix. 4.5 ml of the solution was added to each of the arthrobacter tubes and then poured directly onto plates labeled LIP 11-19-18 PA web. The rest of the solution was poured onto a plate labeled LIP 11-19-18 Control.
6. The plates were allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
7. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

• Bubbles were seen on the control plate

Interpretations/Next Steps:
The procedure was complete. The next step will be to flood the plates and collect the lysate.

Titer Test and Plates Made:

Rationale:  The purpose of this lab is to collect lysate and perform a spot titer test to determine if the lysate is a high titer.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was it.
2. Lysate was harvested from the four flooded plates using a syringe and was filtered into a tube with a 0.22 um syringe filter. Approximately 25 ml were obtained.
3. Serial dilutions were performed out to 10^-5 with the lysate collected(lysate 2) and lysate 1.
4. Top agar was made for one plate, using 2 ml of LB broth, 22.5 ul of CaCl2, 2.5 ml of 2x TA, and 0.5 ml of arthrobacter. The top agar was poured onto a plate labeled LIP 11-16-18 Spot Titer Test. The plate was divided into 7 sections, one for the phage bufer test, and 3 for each of the lysates, 10^-3 to 10^-5.
5. 10 ul of lysate was spotted into each of the six sections, and 10 ul of phage buffer was spotted into its section.
6. The plate was allowed to sit for 15 minutes and then inverted and stored in the incubator until the next lab.
7. The workstation was cleaned and the materials were properly stored or disposed of.

Observations:

• Few bubbles seen on the plate
• Top agar control was made on a second plate by a different group.

Interpretations/Next Steps:
The procedure was complete. The next step will be to determine the titer of the lysate collected and create a TEM grid.

Picture of Plates:

Rationale: The purpose of this lab is to flood four webbed plates to collect more lysate.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit.
2. The four plaque assays done before were flooded with 8 ml of phage buffer each.
3. The plates were stored in the refrigerator until the next lab.

Observations:

• The plates were not completely webbed.

Interpretations/Next Steps:
The procedure was complete. The next step will be to collect the lysate and perform a spot titer test to calculate the titer of the lysate.

Flooded Plates:

Rationale: The purpose of this lab is to calculate the titer of the lysate, flood plates to collect more lysate, and create plaque assays for future flooding.

Description of Procedures: 

1. The workstation was cleaned using aseptic technique and an ethanol burner was lit.
2. The titer was calculated to be 5.1 x 10^7, a medium titer.
3. The 10^-1 and 10^-2 plates were flooded with 8 ml of phage buffer each and placed on the shaker for 1 hour.
4. 10 ul of 10^-2 lysate was added to 4 tubes of 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
5. Top agar was made for 5 plates. 10 ml of LB Broth and 112.5 ul of CaCl2 were added to a tube.
6. 12.5 ml of 2x TA was added to the tube. 4.5 ml of the top agar solution was added to each of the arthrobacter tubes and poured directly onto plates labeled LIP 11-14-18 PA1, LIP 11-14-18 PA2, LIP 11-14-18 PA3, and LIP 11-14-18 PA4. The rest of the solution was poured onto a plate labeled LIP 11-14-18 control.
7. Plates were allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
8. Lysate was harvested from the two flooded plates and filtered with a 0.22 um syringe filter into a tube.
9. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

• PA 3 had many bubbles
• 2 plates flooded, 4 plaque assays made

Interpretations/Next Steps:
The procedure was complete. The next step will be to flood the plates and harvest their lysate, and then calculate the titer.

Serial Dilution Plates and Control:

Rationale: The purpose of this lab is to perform serial dilutions to calculate the titer of the lysate.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an aseptic zone was created with an ethanol burner.
2. Serial dilutions were performed out to a dilution of 10^-5.
3. 10 ul of each dilution was added to 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
4. Top agar was made for six plates. 12 ml of LB and 135 ul of CaCl2 were added to a tube.
5. 15 ml of 2 x TA were added to the tube. 4,5 ml of top agar solution was poured onto a plate labeled LIP 11-12-18 Control. 4.5 ml was mixed with the arthrobacter for each dilution and poured onto five plates labeled LIP 10-12-18 10^-1, LIP 10-12-18 10^-2, LIP 10-12-18 10^- 3, LIP 10-12-18 10^-4 , LIP 10-12-18 10^-5.
6. Plates were allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
7. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

• No 10^0 plate was made.
• Bubbles were seen on all plates.

Interpretations/Next Steps:
The procedure was complete. The next step will be to use the serial dilutions to calculate the titer of the lysate.

Plates: