- [Instructor] In this video,
we're going to walk you through the workflow
for high molecular weight DNA extraction from cells
using the Monarch high molecular weight
DNA extraction kit for cells and blood.
The protocol is broken into two parts,
cell lysis and DNA binding and elution.
In the Monarch workflow,
the cell membranes are first lysed to enable the RNA
in the cytoplasm to be digested by RNase A.
The nuclei containing the genomic DNA remain intact.
If the nuclei were also lysed in this first step
the lysate would become very viscous from the genomic DNA
and this would prevent efficient RNA removal
by the Rnase A enzyme.
After pelleting the cells, prepare the nuclei prep solution
by mixing the nuclei prep buffer
with the Rnase A and prepare the nuclei lysis solution
by mixing the nuclei lysis buffer with the proteinase K.
Flick the tube to resuspend the cell pellet
and add 150 microliters of the nuclei prep solution.
Pipette up and down 10 times to mix
being careful not to introduce air bubbles.
The sample will become less turbid
indicating that the cells are lysing
but the nuclei will remain intact.
Then incubate at room temperature for two minutes.
This nuclei prep process helps reduce the viscosity
of the lysate so that the RNase A treatment
can be effective.
Next, at 150 microliters of nuclei lysis solution
and invert 10 times to mix.
Avoid introducing air bubbles and do not vortex or pipette.
In this step, the nuclei are lysed
releasing the genomic DNA.
Incubate at 56 degrees for 10 minutes in a thermal mixer
with agitation at the desired speed to control the shearing
and tune the size of the gDNA.
The speed of the thermal mixer influences fragment length.
Higher speeds reduce overall size.
For the standard ligation based nanopore sequencing,
agitation at 2000 RPM is recommended.
At 300 RPMs or with no shaking,
maximum fragment length in the megabase range
can be achieved.
These samples will be highly viscous
and difficult to process
but we'll provide the longest possible DNA fragments.
During the incubation, it's helpful to prepare
and label all the plastics that you will need
for part two of the protocol.
After the 10 minute incubation
add 75 microliters of precipitation enhancer
and mix by inverting eight to 10 times.
This solution creates the optimal salt conditions
for the DNA to precipitate and bind to the beads.
Using clean forceps add two DNA capture beads
to each sample, which at this point should be contained
in a Monarch two ML tube.
Add 275 microliters of isopropanol, close the cap
and mix on a vertical rotating mixer
at 10 RPM for four minutes to attach the DNA to the beads.
If you don't have access to a vertical rotating mixer,
you can invert the sample manually 25 to 30 times
slowly and gently.
A manual inversion is complete
when the tube returns to the upright position
and each inversion should take five or six seconds.
If you carry out the inversion slowly enough,
no beads will stick to the bottom of the tube.
After a few inversions, the solution becomes more viscous
and the DNA will wrap loosely around the beads.
During the following inversions,
precipitation of DNA may be visible,
especially with larger sample inputs.
The DNA complex will often contain small air bubbles
and with more inversions,
the DNA will completely wrap around the beads
often causing them to stick together.
Once the DNA is completely wrapped around the beads,
the viscosity of the solution
will drop back to normal levels.
After the inversions are complete,
remove and discard the liquid by pipetting
and there are two ways that you can do this.
The first option is to keep the tube upright
and insert the pipette tip gently pushing the beads aside
to remove the liquid.
The second way is to angle the tube
so that the beads remain at the bottom
and the liquid reaches toward the tube opening.
Then, pipette the liquid from the surface
and continue to tilt the tube as you remove the liquid.
Add 500 microliters of gDNA wash buffer, close the cap
and mix by inverting two to three times.
Then, remove the wash buffer by pipetting
using one of the methods just described.
Repeat this wash step
and once again remove the buffer.
This time you can pour the buffer out
using the pipette tip
to ensure that the bead stay in the tube.
Make sure the bead retainer is inserted
into a collection tube and pour the beads
into the bead retainer.
Discard the used two ML tube.
A quick pulse-spin removes residual wash buffer efficiently
and there is no drying step necessary.
Separate the bead retainer from the collection tube,
discard the collection tube
and pour the beads into a new labeled two ML tube.
Insert the used bead retainer
into a labeled low bind 1.5 ML microfuge tube.
This will be used later for elution.
Add 100 microliters of elution buffer onto the glass beads
and incubate for five minutes at 56 degrees Celsius
in a thermal mixer with agitation at the lowest speed.
Halfway through this incubation,
take the sample out of the incubator
and tilt the tube almost horizontally and shake it gently.
This ensures that the beads can move freely
allowing for complete release of the DNA from the beads.
Place the sample back into the thermal mixer
and finish the incubation.
Make sure that the bead retainer is inserted
into the 1.5 ML microfuge tube.
After the five minute incubation
pour the eluate and the glass beads
into that bead retainer and close the cap.
Centrifuge for 30 seconds to separate the eluate
from the glass beads.
When taking the samples out of the centrifuge,
remove and discard the bead retainer with the beads
and close the microfuge tube containing your eluate.
Before measuring or using your high molecular weight DNA
you'll need to ensure that it's uniformly dispersed.
To do this, first pipette up and down five to 10 times
with a wide bore pipette tip.
Next, you have three options to help the DNA
return to its natural confirmation and solution.
First, you can heat at 37 degrees for 30 minutes to an hour.
Alternatively, you can leave at room temperature overnight.
The third option is to leave at four degrees
for at least 24 hours.
Anytime you wanna quantify your DNA,
it's important to homogenize the sample
by pipetting up and down with a wide bore pipette tip
in order to get accurate measurements.
If your sample is stored at four degrees Celsius,
spin down the sample before pipetting
to collect any droplets that have formed in the tube.
We provide more detailed guidance online
and in the product manual.
If you have any questions or need help with your preps,
our technical support scientists are happy to help.
Contact us at info@neb.com.