Hello, my name is Eric Chow,
and I direct the UCSF Center for Advanced Technology.
In this video, I'll show you how to use magnetic purification beads
-- commonly referred to as AMPure or SPRI beads --
that are commonly used in next-generation sequencing library preparation.
These beads are magnetic particles
that are carboxy coated,
and contain... and are in a solution that contains PEG and sodium chloride.
The PEG and sodium chloride cause nucleic acids
to precipitate out of solution
and use the beads as a nucleation point.
And once they're stuck on the beads,
they can be manipulated with magnets.
What's nice about these SPRI beads
is that they can be tuned
so that you can get different sizes of nucleic acids
binding to the beads by the ratio of this PEG solution that's added.
So, a lot of these purifications
are typically done in PCR tubes
and small magnets like this.
But for the purpose of today's video,
we'll be using Eppendorf tubes, because they're a little bit easier to visualize,
and this larger magnet.
The first step is to take your sample
and add some of the SPRI beads to the sample.
When pipetting the SPRI beads, you want to pipette slowly,
because the solution is very viscous
due to the high PEG concentration.
When you add the mixture to your sample,
you can mix by pipetting up and down,
or you can vortex.
You just want to make sure you get a nice homogeneous suspension.
And if you vortex,
you'll want to give it a quick spin in a benchtop centrifuge,
just to collect all the fluids to the bottom of the tube.
We'll want to let the beads sit, and the solution to sit,
for about five minutes at room temperature
to give the nucleic acids time to bind to the surface of the beads.
So, now five minutes has elapsed,
and we'll put the sample onto the magnetic rack.
Depending on the strength of your magnet
and the volume of beads that you've used,
this can take anywhere from two minutes to five minutes for the beads,
which now contain the nucleic acids,
to move to the back of the tube,
attracted to the magnet.
You'll know this is complete when your solution is clear
and all the beads have been pulled out of solution.
Now, several minutes have elapsed,
and, if you can see,
the beads are now stuck to the back of the tube,
and the nucleic acids that are stuck onto the beads
have also been pulled out of solution.
The next step is to remove the supernatant,
which now contains...
does not contain any of your sample.
When you remove the supernatant,
be careful not to disturb the beads at the back of the tube.
You can discard the supernatant and perform the first wash with 80% ethanol.
Some protocols may call for a different percentage of ethanol,
so just consult your protocol.
When you add the ethanol wash,
you want to be careful not to disturb the pellet.
So, you're not resuspending the beads
or mixing them into solution.
You're simply just adding ethanol to the tube
while the beads are still stuck to the back of the tube.
This is used to wash out any of the residual salt and PEG
present around the beads.
You want to let this sit for 30 seconds.
After 30 seconds have elapsed, remove the first wash.
And then add the second ethanol wash to the tubes,
again making sure you don't disturb the pellets.
30 seconds if elapsed, and we'll remove the second wash.
Try to remove all the residual ethanol from the bottom of the tube
to aid drying.
Now, you'll want to let the beads dry for about five minutes at room temperature.
This allows the excess ethanol that's still present on the beads
to evaporate away.
And depending on the volume of beads that you used
and the size of your tube,
sometimes this drying might happen faster.
During this time, if you notice that the beads
are starting to get dry and not glistening any more,
that's an indication that the beads are fully dry,
and it's ready to go into elution.
So, if you see them starting to turn this...
kind of a dry, cakey brown color,
you'll want to pull 'em off the magnet and go directly to elution.
Five minutes have elapsed,
and now we'll resuspend the beads
with our elution buffer.
Elution buffers simply are just water
or a low concentration of Tris or Tris-EDTA,
so they don't contain high concentrations of salts or PEG.
And this causes DNA that's present on the beads
to resuspend back into solution.
And so, what we'll do is make sure we resuspend the beads
and get rid of any chunks,
and break them up.
And again, you do this by pipetting up and down, or you can vortex.
If you vortex, just make sure to do a quick spin afterwards
to collect the liquid at the bottom of the tube.
And we'll let this sit for 30 seconds to give the nucleic acids
a little bit more time to come off the beads and into solution.
After 30 seconds have elapsed,
place the tube back on the rack
to pull the beads that now do not contain nucleic acids out of solution,
leaving the nucleic acids in your sample in the solution.
So, this second bead-binding step to the magnet
actually occurs a lot faster,
because our elution buffer doesn't contain PEG,
which is very viscous.
So, in the initial separation,
it took several minutes because the high concentration to PEG
slowed the movement of the beads towards the magnets.
And you can see, already, our beads are stuck to the back of the tube,
and we're ready to pull off our eluates.
You want to carefully remove your sample,
and again, not disturb the bead pellet at the back.
If you notice that you've pulled up some beads,
it's okay.
Just go ahead and put your sample back in the tube
and let the sample sit for a few seconds
to allow the beads to stick back to the magnets.
So, now our eluate, with our purified sample,
is now in our new tube,
and we've completed our cleanup.
Thanks for watching.