DNA Extraction Methods

 

GeneAid™ Tissue/Blood DNA Mini Kit (Geneaid Biotech Ltd., Taipei, Taiwan).

  1. Smash Nematode
  2. added to 600 μl of Cell Lysis Buffer (GT buffer from kit)
  3. homogenize
  4. 20 μl of Proteinase K was added to the tube, 
  5. homogenize
  6. incubated at 60°C for 30 minutes.
  7. Every 10 minutes, the tube was shaken to distribute the temperature evenly.
  8. the protein removal step was carried out by adding 200 μl of protein removal buffer (GBT buffer from kit)
  9. homogenized for approximately 10 seconds
  10. incubated at 60°C for 20 minutes  
  11. DNA precipitation step was performed by adding 200 μl of absolute ethanol
  12. The solution was then transferred to the GS column from the kit
  13. centrifuged at 15,000 rpm for 2 minutes.
  14. disregard supernatant
  15. In a new tube, add 400 μl of wash buffer (W1 buffer from the kit) to the pellet
  16. centrifuged at 15,000 rpm for 30 seconds
  17. Centrifugation was repeated at 15,000 rpm for 3 minutes to dry the column matrix. 
  18. The preheated 30 μl elution buffer was inserted into the center of the column matrix  
  19. incubate for at least 5 minutes
  20. centrifuged at 15,000 rpm for 60 seconds.
  21. the suspension in 1.5 ml tubes was ready to be used for the next step or stored at –20°C
  22. (available at: https://geneAid.com/data/files/1605685391109197921.pdf).

 

Cetyltrimethylammonium bromide (CTAB) 2% lysis buffer

  1. Smash Nematode  
  2. added to 200 μl of CTAB buffer solution
  3. incubated at room temperature for an hour
  4. incubated at 65°C for 30 minutes using a water bath,
  5.  shake every 10 minutes to even out the temperature of the suspension.
  6. centrifuged at 2,400 rpm for 5 minutes
  7. homogenize for 1–3 minutes with Chloroform Isoamyl Alcohol (CIAA).
  8. centrifuge the sample at 9,600 rpm for 15 minutes.
  9. the supernatant was separated in a new 1.5 ml Eppendorf tube,
  10. Add cold absolute ethanol (2× the volume of the supernatant)
  11. incubate at –20°C for 24 hours.
  12. centrifuged at 9,600 rpm for 15 minutes
  13. 1 ml of cold 70% alcohol was added to the Eppendorf tube
  14. Inverted tube  
  15. centrifugation of 9,600 rpm for 15 minutes.
  16. disregard the ethanol  
  17. dry pellet for 3 hours.
  18. Add 30 μl of TE solution  
  19. homogenize.
  20. store extracted DNA at –20°C.

 

Sodium dodecyl sulphate (SDS) 1% lysis buffer

  1. Smash Nematode  
  2. added to 1% SDS buffer solution (Mondino et al. (2015)
  3. incubated at 60°C for 30 minutes
  4. transfer supernatant to a new tube
  5. add CIAA solution  
  6. centrifuged at 10,000 rpm for 15 minutes
  7. transfer supernatant to a new tube
  8. Add cold absolute ethanol (2× the volume of the supernatant)
  9. incubated at –20°C for 24 hours
  10. centrifuge at 10,000 rpm for 15 minutes
  11. disregard the ethanol
  12. store DNA pellet
  13. 1 ml of 70% cold alcohol was added to the Eppendorf tube.
  14. invert Eppendorf tube
  15. centrifuged at 10,000 rpm for 15 minutes
  16. disregard the ethanol
  17. dry pellet for 3 hours
  18. add 30 μl of TE solution
  19. homogenize
  20. store extracted DNA at –20°C.

 

 

References  

  1. Optimization of DNA extraction methods for genomic analysis of rice root-knot nematode (Meloidogyne graminicola) using PCR (polymerase chain reaction) and Sanger sequencing
    1. DOI: https://doi.org/10.24425/jppr.2022.144416
  2.