4 Electrophoresis of DNA and RNA Fragments
75
Ethidium bromideisalso commonly added to agarose gels as it allows direct
visualization of DNA. This dye intercalates between the stacked bases and
fluoresces red-orange when exposed to UV light (260- 360 nm). The effect
of ethidium on the mobility oflinear DNA molecules is slight, and its addition to the gel offers the advantage of allowing monitoring of fragment separation during the run. Ethidium bromide can also be used to estimate the
DNA quantity of a fragment by comparing its fluorescence to that of a
known quantity of DNA.
Molecular weight markers
At least one lane containing a ladder of DNA fragments of known sizes is
included on most gels; the choice of markers depends on the range of sizes
to be resolved on the gel. A standard curve can be constructed from the
relative positions of the known markers, and this curve can then be
used to calculate the size of unknown DNA fragments. More often, however,
the size of unknown fragments is simply estimated by comparing the distance they migrate relative to that of known fragments.
Subprotocol 1
The Recovery of DNA Fragments
Frequently, DNA samples are purified by electrophoresis. However, the
samples must somehow be recovered from the gel. Therefore, a number
of methods have been developed to maximize DNA recovery. Regardless
of which method is selected, the purity of the final product critically depends on the quality of the preparative gel. Lane overloading results in significant trapping of other bands within the desired fragment, and re-electrophoresis may be necessary if a highly pure product is desired.
Procedure
Isolation of DNA fragments from agarose gels
In this method, one slices the fragment from a gel, places it in dialysis tubing Electroelution
with buffer and uses electrophoresis to drive the DNA out of the gel slice into
the buffer. The chief advantage of this technique is that it can be used for
recovery of a fragment of any size. The procedure is very inconvenient, how-
75
Ethidium bromideisalso commonly added to agarose gels as it allows direct
visualization of DNA. This dye intercalates between the stacked bases and
fluoresces red-orange when exposed to UV light (260- 360 nm). The effect
of ethidium on the mobility oflinear DNA molecules is slight, and its addition to the gel offers the advantage of allowing monitoring of fragment separation during the run. Ethidium bromide can also be used to estimate the
DNA quantity of a fragment by comparing its fluorescence to that of a
known quantity of DNA.
Molecular weight markers
At least one lane containing a ladder of DNA fragments of known sizes is
included on most gels; the choice of markers depends on the range of sizes
to be resolved on the gel. A standard curve can be constructed from the
relative positions of the known markers, and this curve can then be
used to calculate the size of unknown DNA fragments. More often, however,
the size of unknown fragments is simply estimated by comparing the distance they migrate relative to that of known fragments.
Subprotocol 1
The Recovery of DNA Fragments
Frequently, DNA samples are purified by electrophoresis. However, the
samples must somehow be recovered from the gel. Therefore, a number
of methods have been developed to maximize DNA recovery. Regardless
of which method is selected, the purity of the final product critically depends on the quality of the preparative gel. Lane overloading results in significant trapping of other bands within the desired fragment, and re-electrophoresis may be necessary if a highly pure product is desired.
Procedure
Isolation of DNA fragments from agarose gels
In this method, one slices the fragment from a gel, places it in dialysis tubing Electroelution
with buffer and uses electrophoresis to drive the DNA out of the gel slice into
the buffer. The chief advantage of this technique is that it can be used for
recovery of a fragment of any size. The procedure is very inconvenient, how-