Team:SDU-Denmark/Protocols/Miniprep

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(New page: =Miniprep protocol= We used BIO-RAD Quantum Prep, Plasmid Miniprep Kit. All centrifugation steps are performed at maximum speed (12.000-14.000 g = 14.000 rpm). # Transfer an ON culture...)
 
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=Miniprep protocol=
=Miniprep protocol=

Latest revision as of 10:06, 17 August 2009

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Miniprep protocol

We used BIO-RAD Quantum Prep, Plasmid Miniprep Kit.

All centrifugation steps are performed at maximum speed (12.000-14.000 g = 14.000 rpm).

  1. Transfer an ON culture (2 mL) of plasmid containing cells to a microcentifuge tube. Pellet the cells by centrifugation for 30 sec. Remove all of the supernatant by aspirirating or pipetting.
  2. Add 200 µl of the Cell Resuspension Solution and vortex or pipet up and down until the cell pellet is completely resuspended.
  3. Add 250 µl of the Cell Lysis Solution and mix by gently inverting the capped tube about 10 times (do not vortex). The solution should become viscous and slightly clear if cell lysis has occurred.
  4. Add 250 µl of the Neutralization Solution and mix by gently inverting the capped tube about 10 times (do not vortex). A visible precipitate should form.
  5. Pellet the cell debris for 5 min in a microcentrifuge. A compact white debris pellet will form along the side or at the bottom of the tube. The supernatant (cleared lysate) at this step contains the plasmid DNA.
  6. While waiting for the centrifugation step at step 5, insert a Spin Filter into one of the 2 ml wash tubes supplied with the kit. Mix the Quantum Prep matrix by repeated shaking and inversion of the bottle to insure that it is completely suspended (no tubes are supplied with the sample kit, however, most 2 and 1,5 ml tubes will accommodate the Spin Filters).
  7. Transfer the cleared lysate (supernatant) from step 5 to a Spin Filter, add 200 µl of thoroughly suspended matrix, then pipet up and down to mix. If you have multiple samples, transfer the lysates first, then add matrix and mix. When matrix has been added to all samples and mixed, centrifuge for 30 sec.
  8. Remove the Spin Filter from the 2ml tube, discard the filtrate at the bottom of the tube and replace the filter in the same tube. Add 500µL of Wash Buffer and wash the matrix by centrifugation for 30 seconds.
  9. Remove the Spin Filter from 2 ml tube, discard the filtrate at the bottom of the tube and replace the filter in the same tube. Add 500 µL of Wash buffer and wash the matrix by centrifugation for a full 2 minutes to remove residual traces of ethanol.
  10. Remove the Spin Filter and sicard the microcentrifuge tube. Place the filter in on of the 1.5 mL collection tubes supplied with the kit or ay other standard 1.5 mL microcetrifuge tube which will accommodate the Spil Filter. Add 50 µL of deionized H2O. Elute the DNA by centrifugation for 1 minutes at top speed.
  11. Discard the Spin Filter and store DNA at -20 degress Celsius.
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