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EPICENTRE Forum 5 (1)
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Agarose gel electrophoresis of DNA samples is one of the most common
laboratory procedures. The migration of DNA through agarose gels can
be altered by a number of sample contaminants including the presence
of high levels of protein, especially protein with binding affinity for
DNA. High protein content may result from the original isolation of DNA
from cells or from treatment with modifying enzymes. Here, we describe
a simple and effective method of treating DNA samples containing interfering
levels of protein to allow proper migration of the DNA during electrophoresis.
This method is fast, requires fewer steps than phenol extraction, and
does not result in DNA sample loss.
The method for removing protein contaminants from DNA samples is shown
in Table 1. To demonstrate the use of this technique, two pBR322 DNA
samples were digested with BamH I restriction endonuclease. One
sample was then dephosphorylated using Epicentre's HK™ Thermolabile
Phosphatase according to the protocol supplied with the enzyme. Both
samples were treated with T4 DNA ligase to examine the dephosphorylation
efficiency. Aliquots of each sample were loaded onto a 1% agarose gel
and subjected to electrophoresis in standard TAE buffer. The remainder
of the samples was then treated with 20 µg/ml proteinase K and
0.1% SDS as described in Table 1 and aliquots were subjected to electrophoresis
as above.
Table 1. Treatment of DNA Samples to Remove Contaminating Protein.
- To the DNA sample, add SDS to a final concentration of 0.1%
and proteinase K to a final concentration of 20 µg/ml.
- Incubate the sample at 37°C for 30 minutes.
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Figure 1, Panel A shows the migration of the enzyme-treated DNA samples
before SDS and proteinase K treatment. The plasmid DNA bands separated
poorly and migrated slower than the DNA ladder. The ligation data in
Panel A gives the mistaken impression that the dephosphorylated plasmid
(Lane 3) ligated into concatamers and migrated slower due to its increased
size. Panel B shows the results following treatment with SDS and proteinase
K. The samples migrated correctly in relation to the DNA ladder and each
band is easily distinguishable. This gel clearly shows that the dephosphorylated
plasmid did not ligate, as it is migrating at approximately 4.3 kb, the
size of linearized pBR322 DNA. The ligated linear concatamers in the
non-phosphatase-treated sample (Lane 1) are now easily distinguished
from the linear non-ligated plasmid DNA (Lane 2).
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Figure 1. SDS/proteinase K treatment enables proper migration
of DNA samples in agarose gels. pBR322 samples were digested
with BamH I, and either dephosphorylated, or not, and then
ligated with T4 DNA ligase. The samples were subjected to electrophoresis
in a 1% agarose gel (Panel A) or treated with SDS and proteinase
K as described and subjected to electrophoresis as above (Panel
B). Panel A, Lane 1 and Panel B, Lane 3=kb ladder. |
This quick and easy method for removing contaminating protein from
DNA preparations clearly improves the migration of DNA during electrophoresis
and may simplify the analysis of crude DNA preparations or enzymatically-treated
DNA. Following treatment, the DNA can be directly loaded onto an agarose
gel and subjected to electrophoresis without further purification. This
convenient reaction requires less time and fewer steps than phenol extraction
followed by ethanol precipitation and does not result in DNA sample loss.
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