Rationale:

The purpose of this lab was to familiarize myself with DNA Master with the goal of annotating the genome of the phage NapoleonB.

Procedure/Tools:

• DNA Master

1. The FastA file for the phage Elesar was downloaded.
2. DNA Master was opened and the FastA file for Elesar was selected and opened.
3. An auto-annotation was run for Elesar’s genome.

Results:

The genome of Elesar was auto-annotated revealing 66 genes with 11 reverse genes and 55 forward genes. DNA Master had been previously downloaded and setup.

Conclusion/Future Work:

From the data collected it can be concluded that Elesar has 66 genes, 55 of which are forward and 11 of which are reverse, a greater understanding of DNA Master was also achieved. In the future, I will continue to annotate Elesar in order to become more familiar with DNA Master and the annotation process in order to successfully annotate NapoleonB.

Rationale:

To finish extracting the DNA from the pellet frozen on 11.26.18 and to determine the concentration of the DNA via nanodrop.

Procedures:

1. Defrosted the pellet and added .5mL of sterile water to redissolve the DNA into solution.
2. Added 2mL of 37˙C resin to the vial and mixed.
3. Split the contents of the vial into two microcentrifuge tubes.
4. Centrifuged at 12500g for 3 minutes, removed the supernatant.
5. Added 1mL of 80% isopropanol alcohol and shook to dissolve the pellet.
6. Repeated steps 4-5 two more times.
7. Added 1mL of 80% isopropanol alcohol and shook to dissolve the pellet.
8. Added the contents of both tubes to separate vacuum columns and vacuumed.
9. Dried each column filter by centrifuging for 5 minutes at 12,000g in another microcentrifuge tube.
10. Added 100µL of elution buffer to each filter and transferred to another microcentrifuge tube and centrifuged at 12000g for 1 minute.
11. Combined the contents of both tubes into one new microcentrifuge tube and labeled “NMN 11.28.18 Pippa”.
12. Used 2µL of the DNA to conduct a Nanodrop test

Observations/Data:

When redissolving my pellet several chunks remained undissolved. The nanodrop results were 138.92 for nucleic Acid(ng/uL) with an A280/A260 ratio of 1.922.

Conclusions/Next-Steps:

From the nanodrop test, it can be concluded that I have a fairly high concentration of DNA from the phage “Pippa”, which can now be used to run a gel electrophoresis test this week and to sequence the genome in the future.

Rationale:

To extract from DNA from the phage “Pippa” in order to analyze it and to image my TEM grid with “Pippa” on it.

Procedures:

1. Setup an aseptic zone.
2. Added 10mL of “NMN CEW Lysate 10^0 11.14.18” to a vial labeled “DNA Extraction”
3. Added 40µL Nuclease Mix to the vial and mixed.
4. Added 4mL Phage Precipitant Solution to the same vial.
5. Incubated on shaker at 37˙C for 30 mins.
6. Let sit for 40 minutes at room temp.
7. Centrifuged at 20rpm for 20 minutes.
8. Centrifuged at 7500rpm for 20 minutes.
9. Poured off the supernatant from the pellet and froze the pellet.
10. Imaged the phage “Pippa” via TEM.

Observations:

The vial “DNA Extraction” was erroneously centrifuged at 20rpm for 20 minutes instead of the necessary 7500 rpm for the same amount of time, the proper centrifugation was conducted immediately following the erroneous one. The imaged phage was approximately 26nm  in capsid diameter and 112nm in tail length.

Conclusions/Next Steps:

The next step will be to further prep the pelleted phage for DNA extraction which can then be analyzed for the entire genome of the phage “Pippa”.

Rationale:

To prep a TEM grid with my high titer lysate in order to image the phage it is composed of.

Procedures:

1. Transferred 15µL of NMN CEW Lysate 10^0 to a piece of parafilm in a plate.
2. Transferred 15µL of water onto the same piece of parafilm.
3. Repeated step two for an addtional dot of water.
4. Added one drop of Uranyl Acetate onto the same piece of parafilm.
5. Put a copper mesh TEM disc shiny side down onto the lysate drop for 5 minutes.
6. Transferred the disc to the first drop of water for 2.5 minutes.
7. Transferred the disc to the second drop of water for 2.5 minutes.
8. Transferred the disc to the drop of Uranyl Acetate for 1 minute.
9. Blotted the disc on filter paper and stored for later use with the microscope.

Observations:

There was some questioning regarding if we should have blotted the disc to remove the excess uranyl acetate.

Next Steps:

The next step will be to use the electron microscope to image the phage contained on the copper disc. Additionally, we will also begin the process of prepping the lysate for DNA extraction and analysis.

Rationale:

To create a gridded multi-spot test in order to determine the titer of the lysate produced from the flooding of the plates from 11.12.18.

Procedures:

1. Established an aseptic zone.
2. Filtered the lysate from the four plates flooded by Dr. Adair using a .22µ vacuum filter to produce 10^0 lysate.
3. Diluted 10^0 to create dilutions down to 10^-9 using the procedure of 90µL phage buffer and 10µL of the next highest dilution.
4. In a vial of .5mL of arthro, added 2mL of LB broth and 22.5µL of CaCl2.
5. Added 2.5mL of 2xtop agar and plated on a previously grid labeled plate with 10 boxes for the 10 different dilutions, let solidify 15 minutes.
6. Spotted 5µL of all dilutions onto the gridded plate in their respective boxes.
7. Created a top agar control using 2mL LB broth, 22.5µL of CaCl2, and 2.5mL of 2xtop agar, plated it.
8. Let both plates solidify for 15 minutes then incubated.

Observations:

The plates produced on Monday were all completely lysed as of Tuesday, Dr. Adair proceeded to flood them in order to obtain the phage before it died off, this is what was filtered today. The top agar appeared to be solidifying more quickly than in previous plaque assays and spot tests.

Conclusions/Next Steps:

The next step will be to check the spot test on Friday and from there determine the titer of the lysate that was produced and used to create the spot test. From there it can be determined if the lysate is a high enough titer to be used for TEM and DNA extraction and analysis.

Rationale:

To create a high titer lysate for use in transmission electron microscopy and DNA analysis.

Procedures:

1. Established an aseptic zone.
2. Added 10µL “CEW Lysate” to 90µL phage buffer to make 10^-1 dilution, repeated to make 10^-2 and 10^-3.
3. Added 10µL of each dilution plus 10µL of 10^0 to 4 tubes of arthro, let infect 10 minutes.
4. Added 10mL of LB broth and 112.5µL of CaCl2 to a vial.
5. Added 2mL of this solution to each of the vials of arthro.
6. Added 2.5mL of 2xtop agar to all five vials.
7. Plated all vials in plates following the pattern “NMN 10^x(CEW) 11.12.18”, plated the top agar control.
8. Let solidify and incubated inverted.

Observations:

From the previous plating of this lysate on 11.8.18 it was observed that the entire plate had been lysed and it appeared that arthro had regrown over the plate in a lesser amount, however, it could have also been contamination.

Conclusions/Next Steps:

The next step will be to check on Wednesday to see which plate(s) has been webbed and which are lysed and which are spotted with plaques. We can then use this to determine the titer of the lysate and flood the webbed plate to create a higher titer lysate if needed.

Rationale:

To conduct five more plaque assays with 10^-2 lysate to create five webbed plates that can be flooded to produce a large volume of high titer lysate for later use.

Results from Monday:

Three plates had plaques but not to the extent of a webbed plate. Two plates appeared to be lysed. The top agar control showed some contamination.

.

Procedures:

1. Setup an aseptic zone.
2. Added 20µL of CEW FLE to 180µL of phage buffer to create 10^-1 dilution.
3. Added 20µL of the 10^-1 dilution to 180µL of phage buffer to create 10^-2 dilution.
4. Added 20µL of “10^-2” to a .5mL of arthro, let infect for 15 minutes.
5. Added 2mL of LB broth to two vials, “PA CEW” and “TAC CEW”
6. Added 22.5µL of 1M CaCl2 to both vials.
7. Added infected arthro to “PA CEW”
8. Added 2.5mL of 2xTop Agar to both vials.
9. Shook and plated into respective plates, “CEW 11.7.18 PA” and “CEW 11.7.18 TAC-PA”
10. Let plates solidify for 15 minutes, incubated inverted until Thursday.
11. Created four more “CEW 11.7.18 PA”s using the same procedures listed in steps 4-10.

Observations:

The 10^-2 dilution that was used was freshly made, however, the lysate was fairly old which could have resulted in some degradation of the phage. One of the plate’s top agar’s slid on the plates.

Conclusions/Next Steps:

The next step will be to check to see if the plates are webbed and proceed to flood them with phage buffer if they are webbed.

Rationale:

To conduct five plaque assays with 10^-2 lysate to create five webbed plates that can be flooded to produce a large volume of high titer lysate for later use.

Procedures:

1. Setup an aseptic zone.
2. Added 10µL of “10^-2” to a .5mL of arthro, let infect for 15 minutes.
3. Added 2mL of LB broth to two vials, “PA CEW” and “TAC”
4. Added 22.5µL of 1M CaCl2 to both vials.
5. Added infected arthro to “PA CEW”
6. Added 2.5mL of 2xTop Agar to both vials.
7. Shook and plated into respective plates, “CEW 11.5.18 PA” and “CEW 11.5.18 TAC-PA”
8. Let plates solidify for 15 minutes, incubated inverted until Wednesday.
9. Created four more “CEW 11.5.18 PA”s using the same procedures listed in steps 2-8.

Observations:

The 10^-2 dilution that was used was three days old which could have resulted in some degradation of the phage. Two of the plate’s top agar’s slid on the plates, they were still incubated but right side up. The 4 out of the five tubes of arthro was from a batch of arthro produced in October.

Conclusions/Next Steps:

The next step will be to check to see if the plates are webbed and proceed to flood them with phage buffer if they are webbed.

Rationale:

To conduct a gel electrophoresis test in order to test the effectiveness of the PCR procedures, by testing a PCR sample with a known positive for the phage and seeing if the results of the gel show a positive presence for phage DNA.

Procedures:

1. Added 35mL TBE, 1.7µL EtBr, and .7g Agarose to an Erlenmeyer flask.
2. The flask was microwaved to create a solution.
3. The gel solution was poured into trays and left to solidify, after solidification the dams and comb were removed.
4. The gel apparatus was filled to about 1cm above the actual gel with TBE.
5. Added 5µL of ladder to a well in the apparatus.
6. Added 2.5µL of 10x DNA gel dye to all PCR tubes from Monday.
7. Pipetted 5µL of all PCR tubes into different wells.
8. Attached a power source and ran the test for 40 minutes.
9. Analyzed the gel in the gel imager.

Observations/Data:

The experimental gel came back negative and the control gel performed as expected. The ladder also performed as expected.

Conclusions/Next-Steps:

Based upon the data collected it can be concluded that the PCR procedure is faulty as the gel came back negative for phage DNA when it was known that there was phage DNA in the sample used. The next steps will be to help Claire in producing a high titer lysate that can be imaged with TEM and ultimately sequenced.

Rationale:

To conduct a PCR test in order to test the validity of the PCR procedure used by utilizing lysates that have been confirmed positive.

Procedures:

1. Setup an aseptic zone.
2. Transferred 1mL each of Claire’s ELF-B and Lucy’s ELF-C to micro test tubes, “Claire” and “Lucy” respectively.
3. Boiled both “Claire” and “Lucy”.
4. Added 4.5µL DDI water, 2µL “Claire”, 2µL of “Lucy”, 4µL Primer 1, and 12.5µL of Taq Polymerase to a PCR tube. Labeled with a star and number to indicate Primer type.
5. Repeated step 6 for Primers 2 and 3.
6. Thermocycled all tubes.

Observations/Data:

I observed that some of the additions to the microtubes did not immediately make contact and mix with the main body of fluid.

Conclusions/Next-Steps:

The next step will be to conduct a gel electrophoresis test in order to confirm or deny the validity of the PCR procedure by seeing if the tests come back positive as they are expected to be.