Rationale:

Presented scientific article to the class and set up DNA Master on laptop to use for future genome analysis.

Procedure:

1. Installed and updated DNAMaster on laptop.
2. Downloaded FASTA files for Elesar off phagesdb.org.
3. Performed and analyzed auto-annotation of Elesar.

Observations:

Elesar has 66 genes of which 11 have reverse ORFs and 55 had forward ORFs.

Compared to all of Elesar’s other genes, gene 19 had the longest length which was 4824 nucleotides.

Results:

DNA Master was successful downloaded onto laptop. Also, was able to successfully access ORF Analysis of Elesar as shown below.

Conclusion and Next Steps:

Although my laptop screen looks like gibberish to me now, I look forward to learning and using DNA Master to annotate and perform analyses on NapoleanB. Also, I look forward to improving my presentation skills. I hope to become more familiar with DNA Master by annotating and BLAST Elesar.

Rationale:

Gel electrophoresis was rescheduled for Monday. PCR will be done with the correct concentration of exacted DNA. Since the flash drive carrying the results from the Nanodrop was misplaced, it was decided to redo the Nanodrop readings. Also, more gels were made.

Nanodrop Results:

The results differed greatly from the previous ones. Instead of having a DNA concentration of 888.1 ng/µL, the following values were found.

Nanodrop Results from 11/30

Three trials were performed to confirm that the DNA concentration was much higher than previously recorded. All three trials had the same nucleic acid factor of 50 and the baseline correction of 340 nm. The final trial readings were recorded as Ferranti’s corrected Nanodrop results and were used in PCR calculations.

Procedure:

1. Combined and vortexed 1 µL of exacted DNA (Ferranti) with 9 µL of sterile water to create a Ferranti 10^-1
2. Combined and vortexed 1 µL of Ferranti 10^-1dilution with 9 µL of sterile water to create a Ferranti 10^-2
3. Once an aseptic zone was established, 12.5 µL of 1X MM, 2.2 µL of Ferranti 10^-2dilution, and 6.5 µL of ddH₂O were combined three times into three different microcentrifuge tubes.
4. 4 µL of primer 1, primer 2, and primer 3 were placed into their correlating microcentrifuge tubes.
5. Placed microcentrifuge tubes in thermo-cycler and activated program STU.
6. 40 mL of 1X TAE and 0.8 g of powdered agarose were combined and swirled together in an Erlenmeyer flask.
7. Heated the Erlenmeyer flask until the mixture was boiling then mixed solution until the bubbles disappeared. Repeated until the solution was consistent.
8. Allowed the solution to cool until it was cool enough to touch.
9. Added 2.0 µL of EtBr to the flask to achieve a concentration of 0.5 µg/µL.
10. Poured mixture into gel apparatus and placed comb.
11. Once the gel solidified, the comb was removed, and TAE buffer was poured over the gel to keep it from drying out.

Observations:

• The thermocycler used the program STU which started with 5 minutes of initial denaturation at 98.0ºC. Then, 35 cycles of 30 seconds at 98.0ºC followed by 30 seconds at 55.0ºC followed by 45 seconds at 72.0ºC occurred. After the cycles, a final extension happened at 72.0ºC for 5 minutes.
• A positive control was also made to confirm that the PCR mixture was not contaminated. Anita’s, a phage with a high titer of 2.010⁹pfu/mL, DNA was used.

PCR Calculations:

where

C₁is the extracted DNA concentration (1,121.91 ng/µL)

V₁is the volume needed to add into PCR mixture (needs to be ~ 2 µL)

C₂is the DNA concentration wanted (25 ng/µL)

V₂is the dilution factor

• Steps 6-11 were repeated 4 times to create a total of 4 2% agarose gels.

Next Steps:

Gel electrophoresis will be run with the microcentrifuge tubes prepared from Wednesday (11/28) and the microcentrifuge tubes prepared today so both diluted and non-diluted samples can be compared to each other. Also, these results will identify Ferranti’s cluster.

Rationale:

To find out to which cluster Ferranti belongs, gel electrophoresis will be performed. In preparation for gel electrophoresis, polymerase chain reaction (PCR) will be performed and a 2% agarose gel will be made.

Procedure:

1. Once an aseptic zone was established, 12.5 µL of 1X MM, 2 µL of extracted DNA, and 6.5 µL of ddH₂O were combined three times into three different microcentrifuge tubes.
2. 4 µL of primer 1, primer 2, and primer 3 were placed into their correlating microcentrifuge tubes.
3. Placed microcentrifuge tubes in thermo-cycler and activated program STU.
4. 40 mL of 1X TAE and 0.8 g of powdered agarose were combined and swirled together in an Erlenmeyer flask.
5. Heated the Erlenmeyer flask until the mixture was boiling then mixed solution until the bubbles disappeared. Repeated until the solution was consistent.
6. Allowed the solution to cool until it was cool enough to touch.
7. Added 2.0 µL of EtBr to the flask to achieve a concentration of 0.5 µg/µL.
8. Poured mixture into gel apparatus and placed comb.
9. Once the gel solidified, the comb was removed, and TAE buffer was poured over the gel to keep it from drying out.

Observations:

• The thermocycler used the program STU which started with 5 minutes of initial denaturation at 98.0ºC. Then, 35 cycles of 30 seconds at 98.0ºC followed by 30 seconds at 55.0ºC followed by 45 seconds at 72.0ºC occurred. After the cycles, a final extension happened at 72.0ºC for 5 minutes.
• Unfortunately, it was realized later that DNA concentration was not taken into account when making PCR Mix.
• Also, it was realized later that no controls were made.
• The gel form had 8 wells and had a hazy white tint to it. The following picture shows the gel created.

Next Steps:

Perform gel electrophoresis with DNA samples on agarose gel.

TEM Results:

Unfortunately, the pictures were not of high quality since the copper mesh TEM disc was exposed to uranyl acetate for 15 seconds longer than the protocol instructed, and the disc was bent causing it to begin to deteriorate. The following pictures were taken on the TEM.

Rationale:

Finish DNA extraction protocols so the extracted DNA can be used on the Nanodrop to find the concentration of DNA.

Procedure:

1. Re-suspended phage pellet with 0.5 mL of distilled water.
2. Added 2 mL of 37ºC DNA Clean Up Resin to phage pellet which then was equally distributed into two microcentrifuge tubes.
3. Centrifuged microcentrifuge tubes at 12,500xg for 3 minutes and removed supernatant.
4. Added 1 mL of 80% isopropanol to each microcentrifuge tube and re-suspended pellet.
5. Repeated steps 3 and 4 twice.
6. Added 1 mL of 80% isopropanol to each microcentrifuge tube and re-suspended pellet.
7. Filtered each microcentrifuge tube through one-column syringe.
8. Centrifuged columns at 12,000xg for 5 minutes.
9. Added 50 µL of 80ºC Elution Buffer to each column.
10. Allowed columns to sit for 1 minute before centrifuged at 10,000xg for 1 minute.
11. Performed a blank on the Nanodrop by placing 2.0 µL of Elution Buffer on the sensor, then used 2.0 µL of exacted DNA.

Observations:

• The second phage of which the picture was taken had a head length of 55 nanometers and a tail length of 128 nanometers.
• The pellet that formed after adding the resin and centrifuging was white and cloudy.
• The purified phage genomic DNA was place into a microcentrifuge tube labeled “KEA Ferranti 11/26/18 888.1.”
• Ferranti contained 888.1 ng/µL of nucleic acid.

Rationale:

A TEM grid will be prepared, so a TEM of Ferranti can be taken next time.

Procedure:

1. Placed a slip of parafilm paper inside a petri dish.
2. Pipetted 15 µL of the HTL, 20 µL of DI water twice, and 20 µL of uranyl acetate onto the parafilm.
3. Placed the copper mesh TEM disc in the HTL drop for 5 minutes, then in the DI water drop for 2.5 minutes, then in the other DI water drop for 2.5 minutes, then in the uranyl acetate drop for 1 minute and 15 seconds.
4. Blotted the copper mesh TEM disc with filter paper.
5. Stored the copper mesh TEM disc in A8 in the TEM grid.

Observations:

• The disc was placed shiny side down on all the drops.
• The lysate drop appeared to have a faint crystal goldish color. The DI water drops were clear. The uranyl acetate drop had a faint lime green tint. The picture below shows the drops.

• When placing the copper mesh TEM disc into the A8 spot, the disc crinkled up on itself.

Next Steps:

The TEM grid will be viewed under a TEM. If the TEM does not turn out well, another copper mesh TEM disc will be prepared to view the lysate. Also, the DNA extraction procedures will be completed.

Rationale:

“KEA 11/14/18 Ferranti DNA Ex.” conical vial will be centrifuged and the supernatant will be decanted in order to isolate the phage particles for DNA Extraction.

Procedures:

1. Centrifuged “KEA 11/14/18 Ferranti DNA Ex.” conical vial at 10,000g for 27 minutes at 4ºC.
2. Decanted supernatant from the centrifuged conical vial.
3. Stored pellet in the refrigerator.

Observations:

• At first, the “KEA 11/14/18 Ferranti DNA Ex.” conical vial was centrifuged for only 20 minutes. There was uncertainty whether or not the phage particles separated completely, so the “KEA 11/14/18 Ferranti DNA Ex.” conical vial was centrifuged an additional 7 minutes.
• The supernatant was a light shade of yellow and the phage pellet was a light tannish color. The pellet stuck to the side of the conical vial. The pellet was relatively small. The picture below shows both the phage pellet and supernatant.

Next Steps:

Ferranti will be viewed under a transmission electron microscope. If there is enough time, DNA extraction will be completed.

Results:

No contamination appeared on either the control or spot test plate. The first three dilutions were completely lysed. Plaques formed on the 10^-4to 10^-7dilutions, but not on the 10^-8dilution as shown below.

Rationale:

From the spot plate, the 10^-6dilution will be used to calculate the titer of the lysate. If there is a high titer, some of the lysate will be archived and DNA extraction procedures will begin. If not, more calculations will be performed to determine how much lysate should be used to web a plate.

Procedure:

1. Counted plaques and performed calculations to determine the titer.
2. Once an aseptic zone was established, 2.8 mL of the high titer lysate (HTL) was archived in a conical vial labeled “KEA 11/14/18 Ferranti HTL – 1.3× 10⁹pfu/mL.”
3. 100 µL was placed in microcentrifuge tube labeled “KEA 11/14 TEM.”
4. Diluted the remaining 7 mL of HTL with 3 mL of phage buffer.
5. 40 µL of nuclease and 4 mL of polyethylene glycol (PEG 8000) were combined with the diluted HTL into a conical vial labeled “KEA 11/14/18 Ferranti DNA Ex.”
6. Inverted the conical vial multiple times to mix the solution.
7. Incubated the conical vial for 30 minutes at 37ºC and then at room temperature for 25 minutes.
8. Stored “KEA 11/14/18 Ferranti DNA Ex.” conical vial at 8.2ºC.

Observations:

• 13 plaques were counted under a light microscope as shown in the picture below.

Calculations:

• The titer of the HTL was determined to be 1.3× 10⁹pfu/mL.
• There was barely 10 mL of the HTL. Since DNA extraction requires 10 mL, it was determined that 2.8 mL would be archived, 100 µL would be used for TEM, and the remaining 7 mL would be diluted to 10 mL.
• The titer of the diluted HTL used for DNA extraction was determined to be 9.1× 10⁸pfu/mL.
• After adding the nuclease and PEG 8000, small particles were suspended throughout the lysate.

Next Steps:

Tomorrow, “KEA 11/14/18 Ferranti DNA Ex.” will be centrifuged and the supernatant will be decanted.

Results:

No contamination was found on the plates, and plaques had formed. The “KEA 11/12 10⁰10µL” plate appeared to be the most webbed out of the plaque assays performed on 11/12. The pictures below show these plates.

Rationale:

In all efforts to receive a high titer, the “KEA 11/12 10⁰10µL” plate will be flooded, serial dilutions will be carried out to the 10^-8, and each dilution will be spotted.

Procedure:

1. Once an aseptic zone was established, 8 mL of phage buffer was poured onto “KEA 11/12 10⁰10µL” plate.
2. The plate was shaken on an incubator for five hours.
3. Filtered lysate from flooded plate through a 0.22 µm syringe filter which was added to the “KEA 11/12 FS lysate 10⁰” conical vial.
4. 10 µL of “KEA 11/13 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1dilution which was vortexed.
5. 4 mL of LB Broth, 45 µL of CaCl₂, and 5 mL of 2X TA were combined into a conical vial.
6. 5 mL of the Top Agar (TA) mixture from the conical vial was poured onto a control plate.
7. 5 mL of Arthrobacter was combined with the remaining TA mixture and was poured onto the “KEA 11/13 Spot Test” plate.
8. Once the spot plate had solidified, 10 µL of 10⁰to 10^-8of the “KEA 11/12 FS lysate” and 10 µL of phage buffer were spotted.
9. These plates were placed in the incubator at room temperature.

Observations:

• The following calculations were performed to determine enough LB Broth, 2X TA, and CaCl₂needed for 2 plates.

• Step 4 was repeated with different dilutions to create dilutions out to 10^-8.
• When checking to see if the spots had solidified, it was noted that the spots moved. The spots did not collide with each other; however, the spots were no longer in a circle shape.

Next Steps:

Calculate the titer from the spot test plate.

Results:

Contamination was found on the control plate.

Rationale:

In efforts to receive a high titer, plaque assays will be performed with 400 µL of “KEA 11/5 FS lysate 10⁰,”10 µL of 10⁰and 10 µL of 10^-2of the lysate created on 11/9, 10 µL of 10⁰and 10 µL of 10^-2of the lysate created from flooding and filtering “KEA 11/7 PA 10⁰ 125 µL” plate.

Procedure:

1. Once an aseptic zone was established, 6 mL of phage buffer was poured onto “KEA 11/7 PA 10⁰125 µL” plate.
2. The plate was shaken on an incubator for one hour.
3. Filtered lysate from flooded plate through a 0.22 µm syringe filter into a conical vial labeled “KEA 11/12 FS lysate 10⁰.”
4. 10 µL of “KEA 11/12 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1dilution which was vortexed.
5. 10 µL of “KEA 11/12 10^-1” was added with 90 µL of phage buffer to create 10^-2dilution which was vortexed.
6. 400 µL of “KEA 11/5 FS lysate 10⁰,”10 µL of 10⁰and 10 µL of 10^-2of the lysate created on 11/9, 10 µL of 10⁰and 10 µL of 10^-2of the lysate created on 11/12 were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them.
7. 12 mL of LB Broth, 135 µL of CaCl₂, and 15 mL of 2X TA were combined into a conical vial.
8. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into each test tube.
9. Each test tube was poured onto their correlating plate, and the remaining 4.5 mL of the TA mixture was poured onto a plate labeled “KEA 11/12 Control.”
10. These plates were placed in the incubator at room temperature.

Observations:

• The following calculations were performed to determine the amount of LB Broth, 2X TA, and CaCl₂needed for 6 plates.

• The contamination found on the control plate appeared as small yellow spots as shown below.

• Tiny, microscopic plaques appeared on the plates indicating possibly that the plates were over lysed. The plaques are so small that they do not even appear in photos as shown with the “KEA 11/9 10^-2 10µL” plate below.

Next Steps:

If there is contamination, the experiment will be repeated. The titer will be calculated. If it is a high titer, transmission electron microscopy will take place. If not, webbing calculations and webbing of plates will occur.

Results:

No contamination appeared on the plates, and just as expected no plates were webbed as shown below.

Rationale:

The “KEA 11/7 PA 10⁰150 µL” plate will be flooded and filtered out. Plaque assays will be performed using 400 µL, 450 µL, and 500 µL of the “KEA 11/5 FS lysate 10⁰” and also 15 µL and 10 µL of 10⁰, 10 µL of 10^-1, 10 µL of 10^-2of the new lysate with a goal to receive a webbed plate.

Procedure:

1. Once an aseptic zone was established, 4 mL of phage buffer was poured onto “KEA 11/7 PA 10⁰150 µL” plate.
2. The plate was shaken on an incubator for one hour.
3. Filtered lysate from flooded plate through a 0.22 µm syringe filter into a conical vial labeled “KEA 11/9 FS lysate 10⁰.”
4. 10 µL of “KEA 11/9 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1dilution which was vortexed.
5. 10 µL of “KEA 11/9 10^-1” was added with 90 µL of phage buffer to create 10^-2dilution which was vortexed.
6. 400 µL, 450 µL, 500 µL of “KEA 11/5 FS lysate 10⁰” and 15 µL of 10⁰, 10 µL of 10⁰, 10 µL of 10^-1, and 10 µL of 10^-2of the new lysate were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them.
7. 16 mL of LB Broth, 180 µL of CaCl₂, and 20 mL of 2X TA were combined into a conical vial.
8. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into each test tube.
9. Each test tube was poured onto their correlating plate, and the remaining 4.5 mL of the TA mixture was poured onto a plate labeled “KEA 11/9 Control.”
10. These plates were placed in the incubator at room temperature.

Observations:

• The following calculations were performed to determine enough LB Broth, 2X TA, and CaCl₂needed for 8 plates.

• Air bubbles formed when pouring the plates.

Next Steps:

The best-webbed plate from the plaque assays performed today will be flooded to collect more lysate with a goal to reach a high titer. Also, with the lysate created today, the titer will be calculated. The goal is to have a high titer by Wednesday, the last day to view phages under the transmission electron microscope.