Team:Washington/Protocols/Gel Purification


Gel Purification

  1. Using the QIAquick kit, first excise the DNA fragment form the agarose gel with a clean, sharp blade.
  2. Weigh the gel slice in a tared colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100mg ~ 100µL). For >2% agarose gells, add 6 volumes Buffer QG.
  3. Incubate at 50C for 10 min or until the gel has dissolved completely. Vortex every 2-3 min to help dissolve the gel.
  4. After the gel has dissolved completely, make sure the mixture is yellow (similar to Buffer QG without the dissolved agarose). If the mixture is orange or violet, add 10µL 3M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
  5. Place a QIAquick spin column in a provided 2mL collection tube.
  6. To bind DNA, apply sample to the column and centrifuge for 1 min.
  7. Discard flow-through and place the column back into the same tube.
  8. To wash, add 750µL Buffer PE to the column, let stand for 1 minute, and centrifuge for 1 min.
  9. Discard flow-through and place the column back into the same tube.
  10. Centrifuge the column in the tube again for 1 minute to remove residual wash buffer and discard the collection tube with the flow-through.
  11. Place the column into a clean 1.5mL eppendorf tube.
  12. To elute DNA, add 50uL Buffer EB to the center of the membrane in the column and centrifuge the column in the eppendorf tube for 1 minute. To get a higher DNA concentration, add 30µL Buffer EB to the center of the column membrane, let the column stand for 1min, and centrifuge in the eppendorf for 1 min.To further increase the concentration, letting the column stand for up to 4 minutes after the addition of the Buffer EB can help.
  13. Nanodrop the DNA in the Buffer EB to get the concentration.