Team:JNFLS/Experiments

Protocols

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

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  1. Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. However, it is strongly recommended that fresh TAE buffer or TBE buffer be used as running buffer. Do not reuse running buffer as its pH will increase and reduce yields.
  2. When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
  3. Determine the appropriate volume of the gel slice by weighing it in a clean 15 mL microcentrifuge tube. Assuming a density of 1 g/mL, the volume of gel is derived as follows: a gel slice of mass 0.3 g will have a volume of 0.3 mL
  4. Add1 volume Binding Buffer (XP2).
  5. Incubate at 50-60C for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
  6. Insert a HiBind* DNA Mini Column in a 2 mL Collection Tube.
  7. Add no more than 700 uL DNA/agarose solution from Step5 to the HiBind DNA Mini Column.
  8. Centrifuge at 10，000＊g for 1 minute at room temperature.
  9. Discard the filtrate and reuse collection tube.
  10. Repeat Steps 7-9 until all of the sample has been transferred to the column.
  11. Add 300 uL Binding Buffer (XP2).
  12. Centrifuge at maximum speed (>=13,000 x g) for 1 minute at room temperature.
  13. Discard the filtrate and reuse collection tube.
  14. Add 700 uL SPW Wash Buffer.
  15. Centrifuge at maximum speed for 1 minute at room temperature.
  16. Discard the filtrate and reuse collection tube.
  17. Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix. Note: It is important to dry the HiBind DNA Mini Column matrix before elution.Residual ethanol may interfere with downstream applications.
  18. Transfer the HiBind DNA Mini Column to a clean 15 mL microcentrifuge tube.
  19. Add 15-30 uL Elution Buffer or deionized water directly to the center of the column membrane.
  20. Let sit at room temperature for 2 minutes.
  21. Centrifuge at maximum speed for 1 minute.
  22. Store DNA at -20”C.

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