PEG PRECIPITATION FOR SELECTIVE REMOVAL OF SMALL DNA FRAGMENTS

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PEG PRECIPITATION FOR SELECTIVE REMOVAL OF
SMALL DNA FRAGMENTS



S
         ize-selective precipitation of DNA frag-              presence of short overhangs presumably will not                   James L. Hartley
         ments with polyethylene glycol (PEG) is               affect PEG precipitation.                                         Heather Bowen
         a long-established but underappreciated                                                                                 Molecular Biology
         way of purifying DNA (1,2). Using the                 REFERENCES                                                        Research and
50 bp DNA Ladder as a substrate, the utility of                1. Paithankar, K.R. and Prasad, K.S.N. (1991)                     Development
PEG precipitation in the presence of magnesium                    Nucleic Acids Res. 19, 1346.
                                                                                                                                 Life Technologies, Inc.
is demonstrated.                                               2. Lis, J.T. (1980) Methods Enzymol. 65, 347.
                                                                                                                                 Gaithersburg,
     Aliquots (100 µl) of TE [10 mM Tris-HCl                                                                                     Maryland 20884
(pH 7.5), 1 mM EDTA] containing 1 µg of
50 bp DNA Ladder were added to tubes con-
taining 50 µl of mixtures of 30 mM MgCl2, and                                  A                                   B
PEG 8000 (5, 10, 15, 20, 25, or 30%). After                                     S   1   2    3   4   5   6          1    2   3      4    5   6
mixing, tubes were either centrifuged (10 min at                       bp
room temperature) immediately in a microcen-
trifuge or incubated at room temperature for 10
min before centrifugation. Supernates were com-
pletely removed, and 20 µl TE were added. After                      2,652 —

flicking a few times, 5 µl of agarose gel loading
buffer were added, and 7.5 µl (nominally 300 ng                        800 —
of the ladder) were electrophoresed (figure 1).
     In the presence of 10 mM MgCl2, 10% PEG
                                                                       350 —
(final concentration) did not precipitate the 50-
and 100-bp fragments. At 8.3% PEG, the frag-                           200 —
ments smaller than 200 bp were not recovered.
A final concentration of 6.7% PEG precipitated                          50 —
only those fragments larger than about 650 bp.
Final PEG concentrations of 5% and less did not
precipitate even the 2,652-bp band. Incubation                 FIGURE 1. PEG precipitation of small DNA fragments. Samples were electrophoresed
of the PEG mixtures prior to centrifugation did                in 2% agarose/TAE buffer with 1 µg/ml ethidium bromide at 102 V (6 V/cm) for 35 min
not affect recovery of DNA. Judging from the                   with a cooling fan. Lane S. 250 ng of the 50 bp DNA Ladder. Final PEG concentrations
                                                               were 10%, 8.3%, 6.7%, 5%, 3.3%, and 1.7% in lanes 1–6, respectively. Panel A. Samples were
intensity of the 2,652-bp band, recovery of
                                                               centrifuged immediately after mixing with PEG. Panel B. Samples were incubated for 10
DNA was about 75%. All these fragments had                     min with PEG prior to centrifugation.
blunt ends; however, at room temperature the




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