Extraction of high molecular weight DNA
is important for applications that require
the use of very long DNA molecules,
such as long read sequencing and genome
assembly.
It is often difficult to extract long
DNA that is highly pure and highly
intact. But the Monarch High Molecular
Weight DNA Extraction Kit for Cells and Blood
enables this to be done quickly and effectively.
The Monarch method employs a two
step lysis.
First, the cell membrane is lysed
with a mild nuclei prep buffer mixed
with RNase A.
This releases the contents of the cytoplasm
but leaves the nuclei intact.
The RNA in the cytoplasm is exposed,
allowing it to be efficiently digested
by RNAse A.
Next a mix of nuclei lysis
buffer and proteinase K is added
to the sample, which lyses the nuclei
and releases the genomic DNA.
Proteinase K digests all of the proteins,
including the RNase A enzyme that
has finished digesting the RNA.
This two step lysis approach allows
the RNA to be digested effectively,
before the genomic DNA makes the environment
too viscous, which would significantly
reduce the effectiveness of the RNase A
enzyme.
When working with blood samples the
leukocytes in the blood, which contain
the genomic DNA must first
be isolated.
In mammalian blood, the majority of
cells in blood samples are
erythrocytes, or red blood cells,
which do not contain nuclei but
do contain significant amounts of protein,
mainly hemoglobin.
Removing these red blood cells and their proteins
is very important for a clean blood
prep. Red blood
cells are first lysed with the RBC
Lysis Buffer and their contents
are released into the lysate.
Leukocytes remained mostly unharmed by
the RBC Lysis Buffer,
and they are collected by centrifugation to
separate them from the hemoglobin.
After two wash steps, a clean pellet
of leukocytes can be further processed using
the cell protocol.
During the lysis steps, samples are
agitated using a thermal mixer.
The speed of agitation determines the
fragment length of the DNA.
Low or no agitation produces
very large DNA ranging into
the mega bases, while high speeds
produce short fragments in the range of
50 to 250 kilobases.
Next, alcohol-based precipitation
facilitates attachment of the genomic
DNA to specialized glass beads.
These beads provide a large, smooth
surface to collect the precipitated
DNA and help prevent shearing
during the binding process. High molecular
weight DNA attaches to the beads
and wraps around the large bead surface
during inversion.
Capturing the DNA on the glass beads
surface simplifies the wash
and elution steps, eliminating the
need for lengthy drying steps or multiple
elution steps.
After wash buffer is added to the beads,
the beads with the DNA bound to them can be
poured into the bead retainer, which
holds on to the beads while a short spin
quickly removes all traces of wash buffer
and dries the DNA.
The beads can then be transferred to a new tube
and mixed with elution buffer to begin the
elution process.
After a few minutes of incubation at 56
degrees Celsius,
the DNA slides off of the smooth surface
of the glass beads.
The bead retainer is then used again
to facilitate the separation of the glass beads
from the DNA with a quick and efficient
spin, leaving the user with highly
pure, high molecular weight DNA.
after elution high molecular
weight genomic DNA requires further
manipulation before use and measurement.
The eluted DNA will not be uniformly
dispersed and often clumps in
certain parts of the solution.
Pipetting up and down 5 to 10 times
with a wide bore pipette tip can
help ensure that any visible clumps of DNA
are dispersed into the solution.
Purified DNA samples can then be stored
at four degrees Celsius for future use
or can be prepared for downstream use
as described in the product manual.
We invite you to learn more about this kit
by visiting NEB.com/T3050
where you can find a detailed instruction manual
and other helpful resources.