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For this report we examined the ability of Promega's Wizard® DNA Clean-Up (Cat.# A7280) and Wizard® PCR Preps DNA Purification (Cat.# A7170) Systems to remove ethidium bromide from DNA. Both systems remove ethidium bromide from DNA fragments of various sizes. |
Introduction
Ethidium bromide is a stain commonly used to visualize nucleic acids, particularly in agarose and polyacrylamide gels. However, ethidium bromide intercalates into double-stranded DNA and thus can potentially interfere with downstream applications: removal of ethidium bromide from dsDNA is beneficial, and sometimes essential, to applications involving the use of gel-purified DNA.
Methods
This experiment was conducted using the 1kb DNA Ladder(a) (Cat.# G5711; 250-10,000bp) and a 10mg/ml stock of ethidium bromide (Fisher Scientific, Inc.). The following were mixed together in two separate microcentrifuge tubes: 24µl nuclease-free water, 25µl 1kb DNA Ladder, 1µl of 10mg/ml ethidium bromide stock. These components were incubated at room temperature for 5 minutes to allow the ethidium bromide to intercalate into the DNA, then the total volume (50µl) from one tube was added to either 1ml of Wizard® DNA Clean-Up Resin* or to 1ml of Wizard® PCR Preps Resin*. The DNA was purified following the two recommended protocols (1,2). The resin columns were washed with 2ml of 80% isopropanol and the DNA was eluted with 50µl of nuclease-free water at 65°C. Assuming 100% recovery, the purified and nonpurified DNA should be at the same concentration, since the volumes were identical (50µl before and after purification).
A 10µl aliquot of both the nonpurified and the Wizard® DNA Clean-Up System(a,*) or Wizard® PCR Preps DNA Purification System(a,*) purified DNA was separated on a 1.5% agarose/1X TAE gel. This gel was photographed immediately after electrophoresis using a UV transilluminator (Figure 1, Panel A). The gel was then stained with 0.5µg/ml ethidium bromide for 15 minutes, destained in distilled water for 15 minutes and photographed again (Figure 1, Panel B).
Results
The nonpurified 1kb ethidium bromide-stained DNA can be seen on the agarose gel in lane 1 of Figures 1 and 2. The purified (Wizard® DNA Clean-Up or Wizard® PCR Preps prepared) DNA appears as much lighter bands, suggesting that most or all of the ethidium bromide was removed from the DNA (lanes 2 of Figure 1, Panel A, and Figure 2). Note the intense staining of the upper part of the ladder in lane 1 of Figure 1, Panel A, and Figure 2; free ethidium bromide in the nonpurified sample migrates toward the well, opposite the direction that the DNA migrates.
To verify that the purified and nonpurified samples contained similar amounts of DNA, the gel was stained with ethidium bromide following electrophoresis, then destained with water. As seen in Figures 1 and 2, Panels B, both samples appeared to contain approximately equivalent amounts of DNA (compare lane 1 to lane 2 for Figure 1, Panel B, and lanes 3 and 4 of Figure 2). Thus the light staining of DNA in Figures 1 and 2, Panels A (lanes 2) of the purified sample was due to loss of ethidium bromide and not to a loss of DNA.
Conclusion
This data demonstrates that the Wizard® DNA Clean-Up System and Wizard® PCR Preps DNA Purification System can remove a significant amount of the ethidium bromide from DNA fragments. The ethidium bromide is probably released during the 80% isopropanol wash step. Performing a second wash should remove any remaining ethidium bromide.
References
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Figure 1. Removal of ethidium bromide from DNA using the Wizard® DNA Clean-Up System. Nonpurified (lanes 1) or purified (lanes 2) ethidium bromide-stained 1kb DNA Ladder (Cat.# G5711) was analyzed by electrophoresis on a 1.5% agarose/1X TAE gel. Panel A is a photograph of the gel taken immediately following electrophoresis. To verify the presence of purified DNA, the same gel was stained following electrophoresis with ethidium bromide, then destained with water prior to photography (Panel B). |
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Figure 2. Removal of ethidium bromide from DNA using the Wizard® PCR Preps DNA Purification System. Nonpurified (lanes 1 and 3) or purified (lanes 2 and 4) ethidium bromide-stained 1kb DNA Ladder (Cat.# G5711) was analyzed by gel electrophoresis on a 1.5% agarose/1X TAE gel. Panel A is a gel photograph taken immediately after electrophoresis. To verify the presence of purified DNA, the same gel was stained with ethidium bromide after electrophoresis, then destained with water prior to photography (Panel B). |
