Team:Washington/Protocols/PCR Purification

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Revision as of 03:26, 1 October 2012

PCR Purification

  1. Using the QIAquick kit, add 5 volumes of Buffer PB to 1 volume of the PCR sampe and mix. It is not necessary to remove mineral oil or kerosene.
  2. If pH indicator I has been added to Buffer PB, check that the color of the mixture is yellow. If the mixture is orange or violet, add 10µL of 3M sodium acetate, pH 5.0 and mix. The color of the mixture will turn yellow.
  3. Place a QIAquick spin column in a provided 2mL collection tube.
  4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 minute.
  5. Discard flow-through. Place the QIAquick column back into the same tube.
  6. To wash, add 750µL Buffer PE to the column and centrifuge for 1 minute.
  7. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 minute.
  8. Discard collection tube and place the column in a clean 1.5mL eppendorf tube.
  9. 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.
  10. Nanodrop the DNA in the Buffer EB to get the concentration.
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