Rationale: To create a gel for gel electrophoresis which is then used for imaging along with creating a plaque assay with a known, positive lysate

Procedure:

• Agarose powder (0.80 g) was added to 0.40 mL of 1X TBE then stirred.
• The solution was heated in the microwave until it became clear with no visible particles.
• The flask sat on the lab bench until it became warm to the touch then 2 µL of ethidium bromide with a final concentration of 0.5 µg/µL.
• After stirring the solution, the apparatus was set up with the comb set on the negative side on the apparatus.
• The solution was poured in then remained on the bench for 30 minutes to solidify.
• While solidification, 100 µL of a known, positive lysate was taken and mixed with 0.5 mL of Arthrobacter.
• The top agar was set up by combining 22.5 µL of CaCl2 and 2 mL of LB broth.
• After 10-15 minutes, the lysate and Arthrobacter were added to the calcium chloride and LB broth.
• Then, 2.5 mL of 1X Top Agar was added to the plate and poured onto the plate.
• The plates were left alone to solidify then placed in the incubator.

Results and Analysis:

The results from the imaging came back to negative which may be due to the high presence of ions within the soil sample.

Conclusions:

In order to conduct a gel electrophoresis, the gel must first be created. The agarose powder and TBE were combined in a flask and swirled. Then, the solution was heated to reach a homogenous mixture which was then left alone to cool down to a warmer condition. 2 µL of ethidium bromide was added to the solutions then quickly poured onto the gel apparatus with a comb placed on the side. The apparatus was set aside to solidify for 30 minutes. Then, the lysate and Arthrobacter were mixed together to infect. The LB broth and CaCl2 were added together into a vial of which would become the top agar solution. After 1o minutes, the 2X top agar was added along with the lysate and Arthrobacter onto the plate. It was left alone to solidify then put into the incubator. After solidification of the gel, the comb was removed and TBE was poured to cover the surface of the gel. Four sets of phage buffers were placed, two sets on the outside. The next three sets included samples and the fourth was the control. After the samples, the ladder was filled into the slot. The lid was placed on the power source and was turned to 100 V. The power source remained on. After 40 minutes, the gels were examined.

Future Plans:

If there are plaques on the plaque assay, then two plaques will be picked and diluted for a high titer.