IGEM:Harvard/2007/Protocols/Nucleotide Removal Protocol

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  1. Add 10 volumes of Buffer PN to 1 volume of the reaction sample and mix.
    1. For example, add 500 µl Buffer PN to a 50 µl reaction sample. For DNA fragments ≥100 bp, only 5 volumes of Buffer PN are required.
  2. Place a QIAquick spin column in a provided 2 ml collection tube.
  3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min at 6000 rpm.
  4. For radioactive samples:
    1. Place the QIAquick column into a clean 2 ml collection tube and discard the tube containing the radioactive flow-through appropriately.
  5. For non-radioactive samples:
    1. Discard the flow-through and place QIAquick column back into the same tube. Collection tubes are re-used to reduce plastic waste.
  6. For radioactive samples:
    1. To wash QIAquick column, add 500 µl of Buffer PE and centrifuge for 1 min at 6000 rpm. Discard the flow-through appropriately and repeat wash with another 500 µl of Buffer PE.
  7. For non-radioactive samples:
    1. To wash QIAquick column, add 750 µl of Buffer PE and centrifuge for 1 min at 6000 rpm.
  8. Discard the flow-through and place the QIAquick column back in the same tube, which should be empty. Centrifuge for an additional 1 min at 13,000 rpm (~17,900 x g).
    1. IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifuge.
  9. Place the QIAquick column in a clean 1.5 ml microcentrifuge tube.
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