Purpose
The purpose of this lab was to perform two different, commonly used protocols and compare them. The two protocols were called the Chelex and the Power Soil. Due to the time it takes to perform each protocol, each group at each table performed one of the protocols. These new protocols were performed due to the gel electrophoresis not producing any results of DNA. Although there were no results, we are certain that DNA was available due to the nano drop performed in class by Dr. Adar. It is still uncertain what could have gone wrong to have this negative outcome.

Procedure

Chelex Protocol
• Transfer 300-500 uL dense ciliate culture to a microcentrifuge tube.
• Label the tube and centrifuge it at 6000 x g for 5minutes, and then discard the supernatant.
• Weigh 0.5g of Chelex and transfer 15mL to a conical tube with an addition of 10mL of D.I. water
• Add 200 uL of 5% Chelex to the belle
• Vortex for 1 minute
• Add 15 uL of proteinase K
• Incubate for 30 minutes in 56 degrees Celsius water, and boil for 8 minutes in a 100C water bath or heat block
• Vortex for 1 minute
• Centrifuge at 16,000 x g for 3 min to pellet the cellular debris and Chelex beads
• Remove the supernatant with DNA in a solution to a clean micro centrifuge tube without removing the Chelex beads
• Label the top and side of the microcentrifuge tube and identifiable marker

PowerSoil Protocl
• Add 0.3 grams of soil sample to Power Bead tubes
• Vortex for 5 seconds
• Heat C1 solution to 60C until the precipitate has dissolved
• Add 60 uL of Solution C1 and vortex briefly
• Secure the PowerBead tubes and vortex at a maximum speed for 10min
• Centrifuge the tubes at 10,000 x g for 30 seconds at room temperature
• Transfer the suprernatant to a clean 2mL collection tube
• Add 200 uL of solution C3 and vortex briefly, and then incubate 4 degrees Celsius for 5 minutes
• Centrifuge the tubes at room temperature for 1 minute at 10,000 x g
• Transfer up to 750 uL of the supernatant to a clean 2mL collection tube
• Shake Solution C4
• Add 1.2 mL of solution C4 to the supernatant
• Vortex for 5 minutes
• Load 675 uL onto a spin filter and centrifuge at 10,000 x g for 1 minute at room temperature
• Add 675 uL of supernatant to the spin filter followed by more centrifugation at 10,000 x g for 1 minute at room temperature
• Load remaining supernatant onto the spin filter and centrifuge at 10,000 x g for 1 minute at room temperature e
• Add 50 uL of Solution C5 and centrifuge at room temperature for 30 seconds at 10,000 x g
• Centrifuge at room temperature for 1 minute at 10,000 x g
• Place the filter in a clean 2mL collection tube
• Add 100 uL of solution C6 to the center of the white filter membrane
• Centrifuge at room temperature for 30 seconds at 10,000 x g
• Discard the filter
• Store the DNA in a frozen environment (-20 to -80 C)

Data and Observations

Chelex
Faster to perform compared to the Power soil protocol
DNA is protected by metal ions that stop the enzymatic activity of the nucleases

Powersoil
Produces a more pure sample of DNA making it easier for application and analysis
Beads in Power Bead tubes crush cells to release the DNA

Our group performed the Powersoil Protocol and our final tube was labeled ” 1 TCA C6″
Tube contained the final C6 solution added and stored in freezer

Conclusion and Future steps:
Both protocols took nearly the entire lab period to complete, with the power soil protocol taking a bit longer. In the next lab we will examine the results using a nano drop to observe any DNA samples. If samples are found, we will continue in the amplification process of the DNA found. This time, we will be very careful in not making any mistakes, so we do not have the same results as the Ludox protocol. If the results deem the protocols unsuccessful, we will try again using a positive and negative control.