Rationale:

The goal in lab today was to analyze the spot titer to calculate the titer of the lysate. If the titer calculated was high enough, then the beginning procedure for DNA extraction would begin. If not, then another flood and plaque assay would be run to continue amplifying the titer of the lysate.

Results from 11/14/18:

• Despite being set on the bottom rack of the incubator, which is at a different temperature than the rest of the racks, the spot titer yielded visible plaques up to the 10^-6 dilution. It had completely lysed the first few dilutions, but also contained contamination on the top agar control due to unknown contamination.
• 
• The 10^-6 dilution had approximately 5 plaque forming units (pfu) present, so that was used to calculate the titer of the lysate. Titer was calculated using the equation below.
• 
• Titer was 5*10^8 pfu/mL, which was a high titer lysate and DNA extraction could begin.

Materials:

• High Titer Lysate (HTL)
• Conical Vials
• Nuclease Mix
• Phage Precipitant Solution

Procedure:

1. Established an aseptic zone
2. Lysate had to be archived, and began with naming the phage “Kiersten”.
3. After naming, 2.8 mL of lysate was transferred to a conical vial for archiving.
4. 100 µL of lysate was aliquoted to a micro centrifuge tube for prepping a TEM grid next lab.
5. 10 mL of the HTL was then transferred to a separate 50 mL conical vial, and 40 µL of the nuclease mix was added to the HTL. It was then mixed gently to combine.
6. 4 mL of the phage precipitant solution was added as well, and mixed gently to combine.
7. The lysate solution was then left to sit and incubate at 37 degrees celsius for 3o minutes.
8. Vial was then weighed out and a conical vial was weighed with water to balance out in a centrifuge and the lysate solution was stored in the freezer for the next lab.

Results/Data:

• The lysate was prepped for DNA extraction, but no actual DNA extraction took place. With the high titer of lysate, the increased presence of phage particles will allow for a larger pellet to form, which will aid with DNA extraction.

Conclusions:

The prepping of the HTL allows for the pelleting of phage to occur once run through a centrifuge as the nuclease removes any remaining bacterial DNA floating in the lysate, and the phage precipitant solution pulls water molecules out of the lysate to aid pelleting.

Next Steps:

The next steps for this experiment are to prep a TEM grid for microscopy and pellet the phage particles.