Purifying DNA from Gel Slices

From 2008.igem.org

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'''Back to [[Team:Newcastle University/Notebook]]'''
'''Back to [[Team:Newcastle University/Notebook]]'''
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To purify DNA from a slice of agarose gel we used the GE Healthcare illustra GFX PCR DNA and Gel Band Purification Kit, and the method outlined below is taken from their protocol manual included with the kit.
To purify DNA from a slice of agarose gel we used the GE Healthcare illustra GFX PCR DNA and Gel Band Purification Kit, and the method outlined below is taken from their protocol manual included with the kit.
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'''Protocol for purification of DNA from TAE agarose gel'''
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This can be found [here] http://www5.gelifesciences.com/aptrix/upp00919.nsf/content/EF527125F24048B9C12572BF0081201D?OpenDocument&Path=Catalog&Hometitle=Catalog&entry=1&newrel&LinkParent=C1256FC4003AED40-31B65D2972761A41C125701900490A45_RelatedLinksNew-57DF33EF62AB6478852573DE0066D351&newrel&hidesearchbox=yes&moduleid=39955
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'''1)  Sample Capture'''
'''1)  Sample Capture'''

Revision as of 11:48, 19 September 2008

Back to Team:Newcastle University/Protocols

Back to Team:Newcastle University/Notebook

To purify DNA from a slice of agarose gel we used the GE Healthcare illustra GFX PCR DNA and Gel Band Purification Kit, and the method outlined below is taken from their protocol manual included with the kit.

This can be found [here] http://www5.gelifesciences.com/aptrix/upp00919.nsf/content/EF527125F24048B9C12572BF0081201D?OpenDocument&Path=Catalog&Hometitle=Catalog&entry=1&newrel&LinkParent=C1256FC4003AED40-31B65D2972761A41C125701900490A45_RelatedLinksNew-57DF33EF62AB6478852573DE0066D351&newrel&hidesearchbox=yes&moduleid=39955


1) Sample Capture

  • Weigh a DNase-free 1.5 ml microcentrifuge tube and record the weight.
  • Using a sterilised scalpel blade, long wavelength(365 nm) ultraviolet light and minimal exposure time, cut out the required band, making the band as small as possible.
  • Place agarose gel band into a DNase-free 1.5 ml microcentrifuge tube.
  • Weigh the microcentrifuge tube plus agarose band and calculate the weight of the actual agarose slice.
  • Add 10 μl Capture buffer type 2 for each 10 mg of gel slice.
  • Mix by inversion and incubate at 60°C until the agarose is completely dissolved. Mix by inversion every 3 minutes.
  • place a GFX MicroSpin column into a Collection tube.

2) Sample Binding

  • Centrifuge Capture buffer type 2- sample mix briefly to collect the liquid at the bottom of the tube.
  • Transfer 600 μl Capture buffer type 2- sample mix onto the assembled GFX MicroSpin column and Collection tube.
  • Incubate at room temperature for 1 minute.
  • Spin the assembled column and Collection tube at 16 000 × g for 30 seconds.
  • Discard the flow through by emptying the Collection tube. Place the GFX MicroSpin column back inside the Collection tube.

3) Wash & Dry

  • Add 500 μl Wash buffer type 1 to the GFX MicroSpin column.
  • Spin the assembled column and Collection tube at 16 000 × g for 30 seconds.
  • Discard the Collection tube and transfer the GFX MicroSpin column to a fresh DNase-free 1.5 ml microcentrifuge tube.

4) Elution

  • Add 10–50 μl Elution buffer type 4 OR type 6 to the center of the membrane in the assembled GFX MicroSpin column and sample Collection tube.
  • Incubate the assembled GFX MicroSpin column and sample Collection tube at room temperature for 1 minute.
  • Spin the assembled column and sample Collection tube at 16 000 × g for 1 minute to recover the purified DNA.
  • Proceed to downstream application. Store the purified DNA at -20°C.
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