IGEM:Harvard/2007/week 2

Day 8: Nucleotide Removal (6/25/07)

1. Add 10 volumes of Buffer PN to 1 volume of random library extensions (single primer) and mix.
   1. B1, B2, B3, B4 and S1, S2, S3, S4 (about 50 ul each)
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. Discard the flow-through and place QIAquick column back into the same tube.
5. To wash QIAquick column, add 750 µl of Buffer PE and centrifuge for 1 min at 6000 rpm.
6. 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).
7. Place the QIAquick column in a clean 1.5 ml microcentrifuge tube.
8. To elute DNA, add 100–200 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at 13,000 rpm (~17,900 x g). Alternatively, for increased DNA concentration, add 30–50 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.

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