Rationale:

With DNA extracted and analyzed, it was time to run a PCR and prep a gel to gain some information on the composition of this phage’s genetic material.

Results from 11/26/18:

• From the DNA extracted, there is a very good amount of genetic material present. The DNA contains 805.8 micrograms per ml of nucleic acids, but this could be either phage DNA or bacterial, or both.
• A phage was found intact with a head size of 50 nanometers and a tail size of approximately 174 nanometers.

Materials:

• 3 Microcentrifuge tubes
• Extracted DNA
• Forward Primers and Reverse Primers for clusters (AK, AL, AM, AN, AO, AP, AQ, AR, AS, AT, AU, AV).
• 2X PCR Mix (dNTP, Taq, Buffer, Mg)
• ddH2O
• Thermocycler
• Agarose powder
• TAC
• Microwave
• Ethidium Bromide (DNA dye)
• Erlenmeyer flask
• Electrophoresis apparatus (tray, comb, dams)

Procedure:

1. Established an aseptic zone.
2. Labeled each microcentrifuge tube for their respective primer mixes (1-3).
3. Added 2 µl of extracted DNA to each of the microcentrifuge tubes.
4. Next, added 12.5 µl of the nuclease master mix to each of the tubes.
5. After that, 6.5 µl of ddH2O was added to each of the tubes as a final volume of 25 µl is wanted in each of the tubes.
6. 4 µl of primer mix 1 was added to its respective microcentrifuge tube, then 4 µl of primer mix 2 was added to its respective tube, and the same for primer mix 3 as well.
7. The micro centrifuge tubes were then put on ice to be transferred to the PCR machine, where the “STU” program was selected to run. This included 35 cycles total of 3o seconds at 98.0 °C, 30 seconds at 55.0 °C, 45 seconds of 72 °C, and a final extension of 5 minutes at 72.0 °C.
8. While PCR was running, an agarose gel was prepped by measuring out 0.8 g of agarose powder and combined with 40 ml of TAC in an erlenmeyer flask.
9. The mixture was swirled to combine, and was put in the microwave to boil in one minute increments until it was clear and bubbling. It was then allowed to cool until it was just cool enough to touch without burning ones hand.
10. Next, 2 µl of ethidium bromide was added to yield a final concentration of 0.5% EtBr.
11. The gel was then carefully poured into an assembled electrophoresis apparatus with the comb inserted and left to cool. The comb was then removed and the gel was covered with TAC to prevent any drying to store for the rest of the week.

Results/Data:

• Primer Mix 1 was extremely concentrated when mixed with the DNA sample and could influence the results
• A positive control was not run in the PCR as well.
• DNA was not diluted either, which was needed as the DNA has a large amount of nucleic acids per ml.
• Agarose gel was created with no complications, and the comb was removed without ripping the gel.

Conclusions:

The failure to dilute the DNA or the primer mix will yield interesting potential results. As there is such a high amount of genetic material in the extracted DNA samples, this can cause just a large mass of DNA to be yielded from PCR that will be unable to pass through the gel. PCR should still work theoretically with the highly concentrated primer mix 1, but it is not the most favorable environment for PCR to occur.

Next Steps:

The next steps are to analyze the PCR results and attempt to run a gel with the overly concentrated DNA.