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.

// 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//
 * // NOTE: //**// A dry time of 5 min or more at Room Temperature is optional to ensure all traces of Ethanol //


 * 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