Difference between revisions of "DNA Extraction S. commune QIAgen MagAttract"

From PombEvolution
Jump to navigationJump to search
m
Line 23: Line 23:
 
#Repeat step 16.
 
#Repeat step 16.
 
#Remove the tube holder of the MagAttract Magnetic Rack from its magnetic base and add an appropriate volume of Buffer AE (100–200 μl).  
 
#Remove the tube holder of the MagAttract Magnetic Rack from its magnetic base and add an appropriate volume of Buffer AE (100–200 μl).  
#Place the tube holder onto the mixer and incubate at room temperature for 3 min at 1400 rpm.    ''Optional: Elute the DNA with distilled pure water if the DNA will be used in enzymatic downstream applications.''
+
#Place the tube holder onto the mixer and incubate at room temperature for 3 min at 1400 rpm.    <br>''Optional: Elute the DNA with distilled pure water if the DNA will be used in enzymatic downstream applications.''
#Place the tube holder of the MagAttract Magnetic Rack on the magnetic base, wait until bead separation has been completed (~1 min), and transfer the supernatant with the high-molecular-weight DNA to a new sample tube.  Note: The yield of genomic DNA depends on the sample type and the number of cells in the sample. For some downstream applications, concentrated DNA may be required.  Elution with volumes of less than 200 μl (e.g., 150 μl, 100 μl) increases the DNA concentration in the eluate.   A second elution with Buffer AE increases the total DNA yield. Due to the increased volume, the DNA concentration is reduced.   ''Note: In general, magnetic particles need to be removed before performing downstream applications. Tubes containing eluates should first be placed in the MagAttract Magnetic Rack and the eluates transferred to a clean tube.''
+
#Place the tube holder of the MagAttract Magnetic Rack on the magnetic base, wait until bead separation has been completed (~1 min), and transfer the supernatant with the high-molecular-weight DNA to a new sample tube.  <br>Note: The yield of genomic DNA depends on the sample type and the number of cells in the sample. For some downstream applications, concentrated DNA may be required.  <br>Elution with volumes of less than 200 μl (e.g., 150 μl, 100 μl) increases the DNA concentration in the eluate. <br>  A second elution with Buffer AE increases the total DNA yield. Due to the increased volume, the DNA concentration is reduced.   <br> ''Note: In general, magnetic particles need to be removed before performing downstream applications. Tubes containing eluates should first be placed in the MagAttract Magnetic Rack and the eluates transferred to a clean tube.''
 
[[Category:Schizophyllum]]
 
[[Category:Schizophyllum]]
 
[[Category:DNA extraction]]
 
[[Category:DNA extraction]]

Revision as of 18:12, 9 March 2021

  1. Grind ~20mg freeze dried mycelium to dust
  2. Resuspend the dust into 360µl buffer P1.
  3. Add ~10mg MMX to mixture.
  4. Place the sample in a thermomixer and incubate at 37°C with shaking at 700 rpm for 30 min.
  5. Spin down all debris at 8000 rcf for 5 min at room temperature.
  6. Transfer 180µl supernatant with cut off 200µl pipette to fresh 2ml tube.
  7. Add 20 µl Proteinase K and mix by tapping the tube.
  8. Incubate at 56°C with shaking at 700 rpm for 30 min.
  9. Add 4 μl RNase A (100 mg/ml) and incubate for 2 min at room temperature.
  10. Add 150 μl Buffer AL to the sample. Mix by pulse vortexing.
  11. Add 15 μl MagAttract Suspension G with 140µl Ethanol 96% and 140 μl Buffer MB to the sample. Mix by pulse vortexing.
  12. Transfer the sample tubes to the tube holder of the MagAttract Magnetic Rack.
  13. Place the tube holder of the MagAttract Magnetic Rack onto the mixer and incubate at room temperature for 3 min at 1400 rpm.
  14. Place the tube holder of the MagAttract Magnetic Rack on its magnetic base, wait until bead separation has been completed (~1 min), and remove the supernatant. Note: Avoid disturbing the magnetic bead pellet while aspirating the supernatant.
  15. Remove the supernatant completely.
  16. Add 700 μl Buffer MW1 to the sample and place the tube holder of the MagAttract Magnetic Rack onto the mixer. Incubate at room temperature for 1 min at 1400 rpm.
    • Place the tube holder of the MagAttract Magnetic Rack on its magnetic base, wait until bead separation has been completed (~1 min), and remove the supernatant.
    • Repeat steps 12 and 12a
  17. Add 700 μl Buffer PE to the sample and place the tube holder of the MagAttract Magnetic Rack onto the mixer. Incubate at room temperature for 1 min at 1400 rpm.
    • Place the tube holder of the MagAttract Magnetic Rack on its magnetic base, wait until bead separation has been completed (~1 min), and remove the supernatant.
    • Repeat steps 14 and 14a.
  18. Rinse the particles with 700 μl distilled water while the beads are fixed to the walls of the sample tube. Incubate for 1 min at room temperature and remove the supernatant.
    Important: Do not pipet water directly onto the bead pellet – pipet it into the sample tube against the side facing away from the bead pellet. All pipetting steps must be performed carefully to avoid disturbing the fixed bead pellet (see Figure 2, page 10).
  19. Repeat step 16.
  20. Remove the tube holder of the MagAttract Magnetic Rack from its magnetic base and add an appropriate volume of Buffer AE (100–200 μl).
  21. Place the tube holder onto the mixer and incubate at room temperature for 3 min at 1400 rpm.
    Optional: Elute the DNA with distilled pure water if the DNA will be used in enzymatic downstream applications.
  22. Place the tube holder of the MagAttract Magnetic Rack on the magnetic base, wait until bead separation has been completed (~1 min), and transfer the supernatant with the high-molecular-weight DNA to a new sample tube.
    Note: The yield of genomic DNA depends on the sample type and the number of cells in the sample. For some downstream applications, concentrated DNA may be required.
    Elution with volumes of less than 200 μl (e.g., 150 μl, 100 μl) increases the DNA concentration in the eluate.
    A second elution with Buffer AE increases the total DNA yield. Due to the increased volume, the DNA concentration is reduced.
    Note: In general, magnetic particles need to be removed before performing downstream applications. Tubes containing eluates should first be placed in the MagAttract Magnetic Rack and the eluates transferred to a clean tube.