Team:Aachen/Wetlab/Protocol2

PCR clean-up

  1. Adjust DNA binding condition


    • For very small sample volumes 30 ul adjust the volume of the reaction mixture to 50-100 ul with water. It is not necessary to remove mineral oil.

    • Mix 1 volume of sample with 2 volumes of Buffer NTI (e.g., mix 100 ul PCR reaction and 200 ul Buffer NTI).

    • Note: For removal of small fragments like primer dimers dilutions of Buffer NTI can be used instead of 100% Buffer NTI.

  2. Bind DNA


    • Place a NucleoSpin Gel and PCR Clean-up Column into a Collection Tube (2 ml) and load up to 700 ul sample.

    • Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube.

    • Load remaining sample if necessary and repeat the centrifugation step.

  3. Wash silica membrane


    • Add 700 ul Buffer NT3 to the NucleoSpin Gel and PCR Clean-up Column. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube.

    • Recommended: Repeat previous washing step to minimize chaotropic salt carry-over and improve A(260)/A(230) values.

  4. Dry silica membrane


    • Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.

    • Note: Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal of ethanol can be achieved by incubating the columns for 2-5 min at 70 ℃ prior to elution.

  5. Elute DNA


    • Place the NucleoSpin Gel and PCR Clean-up Column into a new 1,5 mL microentrifuge tube (not provided). Add 15-30 ul Buffer NE and incubate at room temperature (18-25 ℃) for 1 min at 11,000 x g.

    • Note: DNA recovery of larger fragments (>1000 bp) can be increased by multiple elution steps with fresh buffer, heating to 70 ℃ and incubation for 5 min.