March
16
Lab 9: Troubleshooting Methods for DNA extraction/PCR
Purpose: To begin the protocols for MOBIO and Chelex for extracting DNA and to make the control for the MOBIO protocol
Materials:
- D.I. water
- paramecium
- soil sample
- PowerBead Tubes
- Solution C1, C2, C3, C4, C5, C6
Procedure:
- To the PowerBead Tubes provided, add 0.25 g of soil sample (For the control, add 500 ul of the Paramecium culture and continue with the same protocol)
- Gently vortex to mix
- Check Solution C1. If Solution C1 is precipitated, heat solution to 60C until the precipitate has dissolved before use
- Add 60 ul of Solution C1 and invert several times or vortex briefly
- Secure PowerBead Tubes horizontally using the MO BIO Vortex Adapter tube holder for the vortex. Vortex at maximum speed for 10 minutes
- Make sure the PowerBead Tubes rotate freely in your centrifuge without rubbing. Centrifuge tubes at 10,000 x g for 30 seconds at room temperature.
- Transfer the supernatant to a clean 2 ml Collection Tube
- Add 250 ul of Solution C2 and vortex for 5 seconds. Incubate at 4C for 5 minutes
- Centrifuge the tubes at room temperature for 1 minute at 10,000 x g
- Avoiding the pellet, transfer up to 600 ul of supernatant to a clean 2 ml Collection Tube
- Add 200 ul of Solution C3 and vortex briefly. Incubate at 4C for 5 minutes
- Centrifuge the tubes at room temperature for 1 minute at 10,000 x g
- Transfer up to 750 ul of supernatant to a clean 2 ml Collection Tube
- Shake to mix Solution C4 before use. Add 1.2 ml of Solution C4 to the supernatant and vortex for 5 seconds
- Load approximately 675 ul onto a Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature. Discard the filtrate and add an additional 675 ul of supernatant to the Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature. Load the remaining supernatant onto the Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature
- Add 500 ul of Solution C5 and centrifuge at room temperature for 30 seconds at 10,000 x g
- Discard the flow through from the 2 ml Collection Tube
- Centrifuge at room temperature for 1 minute at 10,000 x g
- Carefully place Spin Filter in a clean 2 ml Collection Tube. Avoid splashing any Solution C5 onto the Spin Filter
- 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 Spin Filter. The DNA in the tube is now ready for any downstream application.
Results
The protocols went smoothly. We are trying to find alternate ways of extracting DNA from ciliates since the last protocol turned out to have negative results. The next step in this protocol is extracting DNA with PCR. Each Chelex group made a control sample but only one control sample was made for the MO BIO.