AMPure Purification Protocol

Vasso Makrantoni, Daniel Robertson, Adele L. Marston

Published: 2021-09-22 DOI: 10.17504/protocols.io.bn39mgr6

Abstract

A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.

Before start

Steps

AMPure Purification Protocol

Note

AMPure purification relies on the principle of solid-phase reversible immobilization (SPRI) as previously described [AMPure purification relies on the principle of solid-phase reversible immobilization (SPRI) as previously described [16]. SPRI beads are paramagnetic and coated with carboxyl molecules, which reversibly bind DNA in the presence of polyethylene glycol (PEG) and salt (20% PEG, 2.5 M NaCl mix). PEG causes the negatively charged DNA to bind on the bead surface. This DNA immobilization is dependent on the concentration of PEG and salt in the reaction, and the volumetric ratio of SPRI beads to DNA is critical. Equal volume of beads to DNA will give an SPRI –DNA ratio of one. As this ratio is changed the length of fragments binding and/or left in solution also changes. A lower SPRI–DNA ratio results in larger fragments at elution.]. SPRI beads are paramagnetic and coated with carboxyl molecules, which reversibly bind DNA in the presence of polyethylene glycol (PEG) and salt (20% PEG, 2.5 M NaCl mix). PEG causes the negatively charged DNA to bind on the bead surface. This DNA immobilization is dependent on the concentration of PEG and salt in the reaction, and the volumetric ratio of SPRI beads to DNA is critical. Equal volume of beads to DNA will give an SPRI –DNA ratio of one. As this ratio is changed the length of fragments binding and/or left in solution also changes. A lower SPRI–DNA ratio results in larger fragments at elution.

Equilibrate an aliquot for all purifications needed of AMPure XP beads at Room temperature for 0h 30m 0s before use. Vortex to resuspend.

Pipet carefully the indicated amounts so that no extra beads adhere to the outside of the tip.

Add the AMPure XP beads to DNA in solution and immediately mix thoroughly by repeated pipetting.

Incubate at Room temperature for 0h 10m 0s to allow binding of DNA to beads.

Place on a magnetic rack for 0h 5m 0s.

Remove and discard the supernatant taking great care not to take any beads.

Keep sample on magnetic rack and add 250µL without disturbing the beads.

Incubate for 0h 0m 30s. Remove and discard all supernatant.

10.

Repeat the previous 2 steps once more: Keep sample on magnetic rack and add 250µL without disturbing the beads.

11.

Incubate for 0h 0m 30s. Remove and discard all supernatant.

12.

Let the beads air-dry for 0h 2m 0s–0h 3m 0s at Room temperature.

Note

Overdrying the AMPure beads after the washing step will negatively impact on the DNA recovery. Beads are dry enough as soon as they lose their sheen.

13.

Add the recommended amount of elution buffer (EB from Qiagen kit or ultrapure ddH2O) and resuspend the beads by pipetting.

14.

Incubate at Room temperature for 0h 3m 0s.

15.

Place in magnetic rack for 0h 2m 0s.

16.

Transfer the supernatant to a new DNA LoBind Eppendorf tube.

Note

If, for example, eluting in 30 μl, remove 28 μl very slowly, being careful to prevent bead carryover. If beads are accidentally removed, pipet the sample back into the tube and allow the beads to bind.

AMPure Purification Protocol

Abstract

Before start

Steps

AMPure Purification Protocol

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