- [Instructor] In this video,
we're gonna walk you through the protocol
for high molecular weight DNA extraction
from animal tissues
using the Monarch high molecular weight DNA extraction kit.
This kit enables isolation of very large DNA fragments,
even megabases in size when working with soft organ tissues.
The protocol is broken into two parts,
tissue homogenization and lysis
and DNA binding and elution.
First, you will need to prepare a master mix
of the tissue lysis buffer and proteinase K.
Sample homogenization you can use
either the included microtube pestle
or a rotor stator homogenizer.
When working with soft organ tissues
like liver or kidney, pestle homogenization
allows for the isolation of DNA ranging into the megabases.
Rotor stator homogenization is a convenient solution
but usually will not produce DNA much larger than 250 KB.
First, we'll demonstrate homogenization with a pestle.
Start with the desired amount of tissue in a pestle tube
and make sure to keep fresh samples on ice
and frozen samples on dry ice.
Frozen samples should be thawed briefly
at room temperature first.
To begin the homogenization procedure,
spin down the sample in a benchtop mini-centrifuge
to collect all the tissue material
at the bottom of the tube.
Grind the sample thoroughly using the pestle
to get the thinnest possible layer of tissue.
This will help speed up lysis
and maximize DNA fragment length.
Leave the pestle in the tube.
Use a wide bore pipette tip to add 600 microliters
of the lysis master mix to the sample.
Do not dispose of this tip yet
as it will be used to mix the sample later.
Make sure there is no tissue material left on the pestle.
And if there is, transfer it carefully into the tube
by wiping the pestle tip along the rim of the tube.
Use the wide bore tip to pipette the lysate
up and down a few times.
This ensures all the tissue pieces are lysed efficiently
which is essential for obtaining the highest DNA integrity.
In this example, with mouse kidney powder
you can see that after homogenization
the lysis is very close to being complete.
Discard the pipette tip and immediately place the sample
in the thermo mixer set at 56 degrees Celsius
and at the appropriate agitation speed to begin lysis.
Now we will demonstrate homogenization
using a rotor stator homogenizer.
Make sure your sample is in a compatible two ML tube
and add 600 microliters of the lysis master mix.
Flick or vortex to mix and make sure nothing is stuck
to the walls of the tube.
Insert the tip of the probe into the sample
and homogenize on the lowest setting
until the tissue pieces are no longer visible.
This usually takes five to 10 seconds.
Stop as soon as the lysate begins to foam.
Some optimization may be required
to find the best conditions for your sample type.
Immediately transfer the sample
to a 1.5 ML pestle tube using a wide bore pipette tip
and incubate in the thermal mixer.
Regardless of the homogenization method used,
the sample should be incubated in a thermal mixer
at 56 degrees Celsius for 45 minutes
with agitation at the desired speed.
When working with brain, muscle or any low input samples
stop the agitation after 15 minutes for the best yield.
Finish the incubation without shaking.
When lysis is complete, samples will have changed
from turbid to clear or mostly clear
depending on the tissue type.
The speed of the thermal mixer
influences DNA fragment length and lysis efficiency.
Higher agitation speeds reduced DNA size
and sample lysis time.
For most applications
including ligation based nanopore sequencing
maximum agitation speed is recommended
and will produce DNA fragments predominantly 50 to 250 KB
and ranging up to 500 KB.
For maximum DNA length, agitate at 500 to 700 RPM
while mixing the sample
by manually inverting every 10 to 15 minutes
to optimize the lysis efficiency.
With these low agitation speeds
tissue samples are digested very slowly
and the resulting DNA will be difficult to handle
and more challenging to dissolve after elution.
Once the lysis incubation has finished,
the removal of RNA can be addressed.
At 10 microliters of Rnase A
and invert five to 10 times to mix.
Incubate for 10 minutes at 56 degrees Celsius
at the same agitation speed previously used.
After this incubation, the lysis is complete
but the proteinase K treatment
typically does not remove all of the protein.
Therefore an additional protein removal step is needed
before the DNA can be bound to the beads.
Add 300 microliters of protein separation solution
and invert the sample for one minute.
Centrifuge to the sample for 10 minutes
to create a phase separation.
The sample will separate into a large, clear phase
containing the DNA on top and a smaller often brown phase
containing the protein on the bottom.
For some tissues the protein phase may be green,
yellow or even clear.
Sometimes additional centrifugation time may be needed
to facilitate the phase separation
especially if low agitation speeds were used during lysis.
In the meantime, change the heat block and the thermal mixer
to a two ML block and preheat it to 56 degrees Celsius.
It's also helpful to prepare and label all the plastics
that you'll need for part two during the centrifugation.
The top layer contains the DNA
but a substantial amount of the DNA
will be located close to the interface
between the clear upper phase and the lower protein phase.
Therefore, it's important to transfer
as much of the upper phase as possible
in the upcoming transfer step.
Use a 1000 microliter wide bore tip first
to get most of the sample and transfer it
to a new Monarch two ML tube.
Then, use a 200 microliter wide bore tip
to transfer the rest of the sample.
Here are some additional tips
for the phase separation.
For low input samples,
use a 200 microliter wide-bore tip for the entire transfer.
If a lower phase is not visible,
leave 30 microliters behind
to prevent protein carry over.
For liver samples,
the addition of sodium chloride helps with glycogen removal.
See our detailed guidance in the product manual.
Avoid transferring any material from the protein layer
although one to two microliters will not be detrimental.
If a small amount of the protein phase
enters the pipette tip gently push it back into the tube.
Typically, the transferred volume
will be around 800 microliters or 400 microliters
for low input samples.
If you notice that the volume you've transferred
is significantly lower,
be sure to adjust the volume of isopropanol
in the next step so that it equals 0.7 volumes.
Using clean forceps
add two DNA capture beads to each sample.
Then at 550 microliters of isopropanol, close the cap
and mix on a vertical rotating mixer at 10 RPM
for five 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 around five or six seconds.
If you carry out the inversions 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 beats.
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 GD and a 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 use 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 label 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 elute and the glass beads
into that bead retainer and close the cap.
Centrifuge for 30 seconds to separate the elute
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 elute.
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.