Rationale

Today we will conduct DNA extraction on the precipitate previously made.

Procedure

• Established an aseptic zone.
• 0.5 µL of sterile water was added to the phage pellet to resuspend.
• 2 mL of incubated Resin was added to the vial and was shaken to fully coat the DNA.
• Approximately 1.5 mL of the solution was added to one microcentrifuge tube, and 1.5 mL of the solution was added to another microcentrifuge tube.
• The two tubes were spun at 12-13k g for 3 minutes.
• The supernatant was removed. 1 mL of 80% isopropanol was added to each tube and the pellet was resuspended.
• The two tubes were spun at 12-13k g for 3 minutes.
• The supernatant was removed. 1 mL of 80% isopropanol was added to each tube and the pellet was resuspended.
• The two tubes were spun at 12-13k g for 3 minutes.
• The supernatant was removed. 1 mL of 80% isopropanol was added to each tube and the pellet was resuspended.
• A column syringe was assembled and attached to the vacuum hood. The contents were placed in the column syringe filter and the DNA/resin mixture was trapped in the column filter.
• The two column filters were placed in 2 microcentrifuge tubes and were centrifuged at 12,000g for 5 minutes. This process was repeated twice.
• 100 µL of Elution Buffer was added to the column and was set aside for 1 minute. The two microcentrifuge tubes were centrifuged at 12,000 g for 1 minute. The product of the two tubes were combined and were placed into the -20 C freezer as a result.

Observation

Dr. Adair centrifuged the phage precipitate preparation before lab since the centrifuging swinging bucket was not functioning at the previous lab.

Conclusions/Next Step

Next, the DNA will be analyzed using a Nanodrop and PCR will occur.