4 Electrophoresis of DNA and RNA Fragments
77
Gelslicesare dissolved when incubated with this enzyme. This is particularly useful when one is working with very small quantities of a fragment.
This protocol is especially useful in purifying (without shearing) high molecular weight fragments sliced from pulsed field gel electrophoresis.
The number of methods that are commercially available increases each
year. The two most commonly used systems, Gene Clean (Bio 101) and
Prep-a-Gene (BioRad), differ primarily in the buffer they use to dissolve
the agarase slice. Gene Clean (Bio 101) uses Nal whereas Prep-a-Gene
(BioRad) uses Na perchlorate. Both bind the DNA to a silicon-based matrix
which is then washed prior to DNA elution. Both produce yields of up to
80% when used for recovering fragments from 0.2-16 kb in size.
Isolation of DNA fragments from polyacrylamide gels
Purifying DNA fragments from polyacrylamide gels is much simpler than
from agarose. The DNA is eluted from the slice by simply incubating the
sample in 0.5 M ammoniumacetateil mM EDTA buffer.
Subprotocol 2
Electrophoresis of RNA
Most commonly, RNA is electrophoresed for Northern blotting. The size
and amount of a specific RNA molecule are then determined by hybridization of a labeled cDNA or anti-sense RNA probe to the blot. The quality of an
RNA preparation can also be assessed by electrophoresis. For intact RNA
samples, two sharp bands (28S and 18S ribosomal RNAs) should be readily
visible when the gel is stained with ethidium bromide and exposed to UV
light.
Many of the general principles discussed previously concerning DNA are
applicable to the analysis of RNA. The migrationrate of RNA molecules
electrophoresed through agarase gelsalso is inversely related to the logarithm of the molecular weight. The size of unknown molecules can be determined by comparing their migration rates relative to those of RNA markers of known size, which are commercially available. The 28S and 18S
bands are 5.0 kb and 1.9 kb respectively in humans, and are also useful
for estimating size. A distinct feature of RNA molecules, however, is
that they have considerable secondary structure since they are singleAgarose
Silicon-based
(glass-milk) purification systems
77
Gelslicesare dissolved when incubated with this enzyme. This is particularly useful when one is working with very small quantities of a fragment.
This protocol is especially useful in purifying (without shearing) high molecular weight fragments sliced from pulsed field gel electrophoresis.
The number of methods that are commercially available increases each
year. The two most commonly used systems, Gene Clean (Bio 101) and
Prep-a-Gene (BioRad), differ primarily in the buffer they use to dissolve
the agarase slice. Gene Clean (Bio 101) uses Nal whereas Prep-a-Gene
(BioRad) uses Na perchlorate. Both bind the DNA to a silicon-based matrix
which is then washed prior to DNA elution. Both produce yields of up to
80% when used for recovering fragments from 0.2-16 kb in size.
Isolation of DNA fragments from polyacrylamide gels
Purifying DNA fragments from polyacrylamide gels is much simpler than
from agarose. The DNA is eluted from the slice by simply incubating the
sample in 0.5 M ammoniumacetateil mM EDTA buffer.
Subprotocol 2
Electrophoresis of RNA
Most commonly, RNA is electrophoresed for Northern blotting. The size
and amount of a specific RNA molecule are then determined by hybridization of a labeled cDNA or anti-sense RNA probe to the blot. The quality of an
RNA preparation can also be assessed by electrophoresis. For intact RNA
samples, two sharp bands (28S and 18S ribosomal RNAs) should be readily
visible when the gel is stained with ethidium bromide and exposed to UV
light.
Many of the general principles discussed previously concerning DNA are
applicable to the analysis of RNA. The migrationrate of RNA molecules
electrophoresed through agarase gelsalso is inversely related to the logarithm of the molecular weight. The size of unknown molecules can be determined by comparing their migration rates relative to those of RNA markers of known size, which are commercially available. The 28S and 18S
bands are 5.0 kb and 1.9 kb respectively in humans, and are also useful
for estimating size. A distinct feature of RNA molecules, however, is
that they have considerable secondary structure since they are singleAgarose
Silicon-based
(glass-milk) purification systems