Agencourt AMPure XP PCR Purification Protocol

2014-08-29

azim58 - Agencourt AMPure XP PCR Purification Protocol

This is the most current protocol I found on the company's website.
"C:\kurt\storage\CIM Research
Folder\DR\2013\2-2-13\data_download\AMPureXP Protocol PRC
purification.pdf"

AMPure Process Overview
Notes from manual
AMPure XP utilizes an optimized buffer to selectively bind PCR amplicons
100bp and larger to paramagnetic beads.
Excess primers, nucleotides, salts, and enzymes can be removed using a
simple washing procedure. The resulting purified PCR product is
essentially free of contaminants.

Materials
AgenCourt Ampure XP last seen on Felicia's 4 C shelf
Magnetic plate last seen in Felicia's drawer

AMPure DNA Purification Protocol (96 well format) 6/24/11

1. Determine whether or not a plate transfer is necessary.

If the PCR reaction volume multiplied by 2.8 exceeds the volume of the
PCR plate, a transfer
to a 300 μL round bottom plate is required..
-Note that regular 200 uL PCR tubes can be used with the magnetic plate

2. Gently shake the Agencourt AMPure XP bottle to resuspend any magnetic
particles that may have settled. Add Agencourt AMPure XP according to the
PCR reaction volume chart below:

PCR Reaction Volume (μL)
 AMPure XP Volume (μL)

10
 18

20
 36

50
 90

100
 180

The volume of Agencourt AMPure XP for a given reaction can be derived
from the following
equation: (Volume of Agencourt AMPure XP per reaction) = 1.8 x (PCR
Reaction Volume)

3. Mix reagent and PCR reaction thoroughly by pipette mixing 10 times.
Let the mixed samples incubate for 5 minutes at room temperature for
maximum recovery.

This step binds PCR products 100bp and larger to the magnetic beads.
Pipette mixing is
preferable as it tends to be more reproducible. The color of the mixture
should appear
homogenous after mixing.

4. Place the reaction plate onto an Agencourt SPRIPlate 96 Super Magnet
Plate for 2 minutes to separate beads from the solution.

Wait for the solution to clear before proceeding to the next step.

5. Carefully remove with a pipette the cleared solution from the reaction
plate and discard.

This step must be performed while the reaction plate is situated on the
Agencourt SPRIPlate
96 Super Magnet Plate. Do not disturb the ring of separated magnetic
beads. If beads are
drawn out, leave a few microliters of supernatant behind.

6. Dispense 200 μL of (fresh) 70% ethanol to each well of the reaction
plate and incubate for 30 seconds at room temperature. Carefully remove
with a pipette the ethanol and discard. Repeat for a total of two washes.

It is important to perform these steps with the reaction plate situated
on an Agencourt
SPRIPlate 96 Super Magnet Plate. Do not disturb the separated magnetic
beads. Be sure to
remove all of the ethanol from the bottom of the well as it is a known
PCR inhibitor.

NOTE: A dry time of 5 min or more at Room Temperature is optional to
ensure all traces of Ethanol
are removed but take care not to over dry the bead ring (bead ring
appears cracked) as this
will significantly decrease elution efficiency.
Note: According to the manual, 70% ethanol is hygroscopic (which means
that it tends to absorb moisture from the air). Fresh 70% ethanol should
be
prepared for optimal results

7. Off the magnet plate, add 40 μL of reagent grade water to each well
of the reaction plate and pipette mix 10 times.

The liquid level will be high enough to contact the magnetic beads at a
40 μL elution volume.
A greater volume of elution buffer can be used, but using less than 40
μL will require extra
mixing (to ensure the liquid comes into contact with the beads) and may
not be sufficient to
elute the entire PCR product. Elution is quite rapid and it is not
necessary for the beads to go
back into solution for it to occur.

8. After mixing, let sit on bench for 2 minutes.

9. Place the reaction plate onto an Agencourt SPRIPlate 96 Super Magnet
Plate for 1 minute to separate beads from the solution.

10. Transfer the eluant to a new plate.

-When nanodropping sample or transferring sample, place the tube onto the
magnetic plate to exclude any remaining magnetic beads leftover.

For long term freezer storage, Agencourt recommends transferring
Agencourt AMPure XP purified samples into a new plate to prevent beads
from shattering.

------------

Miscellaneous Notes recorded 8-28-12
old note before 3-1-13
-Instances of reagent
Agencourt Ampure XP 3-12-13

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