Lab 9 Powersoil and Chelex
3/15/18
Today in lab our group did the Powersoil procedure and the other group did the Chelex procedure. In both procedures the goal was to extract DNA. We did 2 different protocols to test which one yields better results. We are tried different protocols because no DNA appeared in the gel electrophoresis
Procedures
Powersoil
- Obtain a PowerBead Tube and add 0.25 grams of dry soil
- Gentry vortex to mix
- Add 60 µL of Solution C1 and vortex briefly
- Secure the PowerBead Tubes horizontally using the MO BIO Vortex and vortex for 10 minutes at maximum speed
- Centrifuge the tube at 10,000 g for 30 seconds
- Transfer all the supernatant from the PowerBead Tube to a clean 2 mL collection tube
- Add 250 µL of Solution C2 and vortex for 5 seconds
- Incubate for 5 minutes at 4°C in the refrigerator
- Centrifuge the tube at 10,000 g for 1 minute
- Transfer up to 600 µL of the supernatant to a clean 2 mL collection tube; make sure to avoid the pellet when doing so
- Add 200 µL of Solution C3 and vortex briefly
- Incubate for 5 minutes at 4°C in the refrigerator
- Centrifuge the tube at 10,000 g for 1 minute
- Transfer up to 750 µL of the supernatant to a clean 2 mL collection tube
- Add 1.2 mL of Solution C4 to the supernatant and vortex for 5 seconds
- Obtain a Spin Filter
- Load 675 µL of the solution into the spin filter and centrifuge at 10,000 g for 1 minute
- Repeat that step until all the solution is gone, but keep the spin filter
- Add 500 µL of Solution C5 to the spin filter
- Centrifuge at 10,000 g for 30 seconds
- Discard the flow through from the 2 mL collection tube
- Centrifuge at 10,000 g for 1 minute to dry and remove the remaining Solution C5
- Carefully remove the spin filter and make sure no flow through splashes it
- Place the spin filter into a clean collection tube
- Add 100 µL of Solution C6 to the center of the spin filter membrane
- Centrifuge at 10,000 g for 30 seconds
- Discard the SPIN FILTER and keep the filtrate
- Label and store DNA (-20°C to -80°C)
Chelex
- Transfer 300-500 µL of dense ciliate culture (Paramecium culture) to a micro centrifuge tube
- Label the tube
- Centrifuge at 6,000 g for 5 minutes
- Discard the supernatant
- Weigh 0.5g Chelex and transfer to a 15 mL conical tube
- Add 10 mL of DI water
- Add 200 µL of 5% Chelex to the pellet
- Vortex for 1 minute
- Add 15 µL of proteinase K
- Incubate for 30 minutes in 56oC heat block
- Boil for 8 minutes in 100oC heat block
- Vortex for 1 minute
- Centrifuge at 16,ooo g for 3 minutes
- Transfer supernatant with DNA in solution to clean micro centrifuge tube
- Label and store DNA
Observations
0.3 g of soil
Thoughts
On the last step of the powersoil procedure, our group accidentally disposed of the liquid instead of the filter and ended up throwing out our DNA. [I know we messed up pretty bad 🙁 ] So Dr. Adair told us to run 100 uL more of the C6 through the filter again to hopefully get some DNA out but our DNA concentration will probably be much lower than the other groups if we even have any DNA. We labeled our tube 8.