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Recovery of PCR Products from Gels 77
18
Technical Notes for the Recovery and Purification
of PCR Products from Acrylamide Gels
David Stirling
1. Introduction
Although the best way of obtaining pure polymerase chain reaction (PCR) product
will always be to optimize reaction conditions to yield only one product, there are
still circumstances where DNA has to purified from gels. Several good commercial
products exist for the recovery of DNA from agarose. Here, we present a reliable
method of recovering DNA from polyacrylamide gels.
2. Materials
1. Sterile scalpel blade.
2. Microfuge tubes (0.5 and 1.5µL).
3. Sterile narrow-gauge needle (23-guage).
4. Tris-HCl EDTA buffer: 10 mM Tris-HCl, 10 mM EDTA, pH 8.0.
5. 3 M sodium acetate (pH 4.0).
6. 100% ethanol.
7. 70% ethanol.
3. Method
1. Stain the gel with ethidium bromide, visualize the band of interest, and dissect with
scalpel blade.
2. Pierce hole in the bottom of a 0.5 ml Microfuge tube with narrow gauge needle.
3. Place 0.5 mL of TE in a 1.5-mL microfuge tube.
4. Place the acrylamide slice with the dissected DNA band into a 0.5-mL tube (with hole) and
place inside the 1.5-mL tube (the lip will prevent it touching the bottom).
5. Microfuge the two-tube combination at 12,000g for 5 min. The acrylamide will be forced
through the hole in the bottom of the 0.5-mL tube into the TE.
6. Discard the empty 0.5-mL tube.
7. Cap the 1.5-mL tube, vortex for 30 s, then incubate for 2 h overnight at 37°C.
8. Microfuge at 12,000g for 5 min.
9. Transfer supernatant to fresh tube.
10. Add 1/10 volume 3 M sodium acetate and 2 volumes 100% ethanol.
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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