Jan 04, 2016
Titration of AmPure XP Beads for Removal of Fragments
- Matthew Sullivan1
- 1Matthew Sullivan Lab, University of Arizona/Ohio State University
- VERVE Net
- Sullivan Lab
Protocol Citation: Matthew Sullivan 2016. Titration of AmPure XP Beads for Removal of Fragments <400bp. protocols.io https://dx.doi.org/10.17504/protocols.io.c52y8d
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: June 23, 2015
Last Modified: March 15, 2018
Protocol Integer ID: 922
Guidelines
Note: AmPure XP beads are normally used at 80:100 ratio* (beads:DNA) to remove fragments less than 100bp and retain all DNA above 100bp. If you lower that ratio. then DNA will not bind to the beads as efficiently (due to decreased amount of PEG present) and you will lose some of the DNA. To recover all DNA, make sure the starting DNA is not too concentrated and hold onto the residual sample after beads first adhere to magnet; in this way you can re-bind any unbound DNA to fresh beads.
*see step 17 annotation for more info
Agencourt® AmPure® XP beads: Beckman Coulter #A63880
100bp DNA ladder: New England Biolabs (NEB) #N3231S (comes with 6xGLB)
6x Blue Orange Loading Dye: Promega #G190A (optional)
Magnetic Particle Concentrator (MPC): Invitrogen Dyna-Mag2 #123-21D
Materials
MATERIALS
Agencourt AmPure XP beadsCatalog #A63880
100 bp DNA Ladder - 100 gel lanesCatalog #N3231S
Blue/Orange Loading Dye, 6XCatalog #G190A
Magnetic Particle Concentrator (MPC): Dyna-Mag2Catalog #12321D
AmPure XP Bead Titration
AmPure XP Bead Titration
Label 11x1.5 ml tubes 40-45-50-55 etc. to 90 (= ratio of beads to DNA)
Mix 48μl NEB 100bp ladder with 1152μl Molecular Biology Water.
Pipet 100μl of mixture into each of the 11 tubes.
Vortex bottle of AmPure XP beads and dispense 800μl into fresh tube.
Using same pipettor used for step #3, pipet volume of beads into each tube (i.e., 40μl into tube labled 40, 45μl into tube labed 45, etc.)
Close lids, vortex tubes and incubate 5 minutes at room temperature.
00:05:00
Place tubes in MPC rack; wait 5 minutes for beads to adhere to magnet side of tubes.
00:05:00
Pipet off supers and discard them.
Wash the beads 2x with 500μl each 70% ethanol with 30 second incubation.
00:00:30
Remove all supers from tubes.
Place tubes (with caps open) into 37°C heat block to dry beads (evaporate residual ethanol).
Add 10μl Qiagen EB to each tube, close lids.
Vortex to resuspend beads.
Place tubes back into MPC rack and let beads adhere to magnet sides of tubes.
Pipet off supers (containing eluted DNA) into fresh tubes.
Run samples in 2% agarose gel (1x TAE) using 5μl DNA and 1μl 6x gel loading buffer.
Photograph results and determine what ratio of beads to DNA to use to exclude lower range of DNA.
Note
AmPure XP beads are normally used at 80:100 ratio (beads:DNA) to remove fragments less than 100bp and retain all DNA above 100bp. If you lower that ratio, then DNA will not bind to the beads as efficiently (due to decreased amount of PEG present) and you will lose some of the DNA. To recover all DNA, make sure the starting DNA is not too concentrated and hold onto the residual sample after beads first adhere to magnet; in this way, you can re-bind any unbound DNA to fresh beads.