PCR normalization and size selection with magnetic beads

Dominik Buchner

Published: 2022-10-11 DOI: 10.17504/protocols.io.q26g7y859gwz/v1

Abstract

This protocol describes how to clean up and normalize PCR products or DNA extracts and perform a size selection with carboxylated-magnetic beads and a PEG-NaCl buffer. It works by diluting the beads so that the binding capacity is lower than the PCR yield which leads to a normalization of all PCR products to the binding capacity.

Before start

Make sure all buffers are prepared before starting.

For easier pipetting let the normalization solution adjust to Room temperature.

Note

The protocol described here is designed for the use of 250µL, but can also be done in tubes, PCR plates, strips, or any sufficient reaction vessel. The recommended shaking speeds are adjusted to the plates mentioned in the materials.

Steps

Shake the normalization solution until the beads are homogeneously resuspended

Note

The protocol described here uses a normalization solution to sample ratio of 0.7:1. This is sufficient for the removal of primer and primer dimers below a size of 200 bp. For the removal of shorter or larger fragments, the ratio has to be adjusted accordingly. For more information on ratios refer to the material provided in the tab "Guidelines".

Note

The protocol described here is designed for 9µL . If the PCR assay is larger, less water has to be added in step two. It's recommended to keep the amount of normalization solution as is to achieve an output concentration of about 2ng/µL .

Add 31µL and 28µL to a 250 µL U-bottom assay plate

Note

It's recommended to increase the volume of the sample with PCR-grade water for easier liquid handling but also to lower relative pipetting error (e.g. if the pipette is off by 2µL the effect on the ratio is larger if working with a 10µL assay than when working with a 80µL assay.The amount of beads is calculated as follows:(sample volume + water volume) * ratio = cleanup solution volumeIn this example:(9 µL PCR product +30 µL PCR-grade water ) * 0.7 = 28 µL cleanup solutionFor higher sample numbers PCR-grade water and cleanup solution can be prepared as a master mix.

Add 9µL

To bind the DNA to the beads shake at 900rpm

Note

If the protocol is not done in plates mixing can also be accomplished by pipetting or vortexing.

Place the plate on a magnet to pellet the beads for 0h 2m 0s

Note

The bead pellet might be barely visible at this point.

Note

Depending on the magnet and volume used separation times may vary and have to be adjusted accordingly.

Discard the supernatant by pipetting

With the plate still on the magnet, add 100µL to each sample

Incubate for at least 0h 0m 30s

Discard the supernatant by pipetting

10.

11.

With the plate still on the magnet, incubate the plate for 0h 5m 0s at Room temperature to dry off residuals of wash buffer

12.

Add 50µL of elution buffer to each sample

13.

900rpm

14.

Place the plate on a magnet to pellet the beads for 0h 2m 0s

Note

The bead pellet might be barely visible at this point.

15.

Transfer 40µL of the DNA to a new PCR plate. Store at -20°C

Note

Leaving 10µL of elution buffer is recommended to avoid carry-over of beads. If all of the DNA is needed for subsequent analysis try to pipette slowly without disturbing the pellet.

PCR normalization and size selection with magnetic beads

Abstract

Before start

Steps

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