Oct 19, 2022

Public workspacePCR cleanup and size selection with magnetic beads V.3

DOI
dx.doi.org/10.17504/protocols.io.36wgqj45xvk5/v3
  • 1University of Duisburg-Essen, Aquatic Ecosystem Research
Dominik Buchner
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DOI: dx.doi.org/10.17504/protocols.io.36wgqj45xvk5/v3
Protocol Citation: Dominik Buchner 2022. PCR cleanup and size selection with magnetic beads. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqj45xvk5/v3Version created by Dominik Buchner
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
We use this protocol and it's working
Created: October 19, 2022
Last Modified: October 19, 2022
Protocol Integer ID: 71537
Keywords: pcr cleanup, carboxylated beads, magnetic beads, PEG-NaCl precipitation, size selection, buffer exchange
Abstract
This protocol describes how to clean up PCR products or DNA extracts and perform a size selection with carboxylated-magnetic beads and a PEG-NaCl buffer. It can also be used for volume reduction of a sample or for buffer exchange.


Guidelines
Follow general lab etiquette. Wear gloves to prevent contaminating the samples. Clean the workspace before starting with 80% EtOH.

Ratio Guide:
To get an estimate the performance of different ratios the protocol was tested using a DNA Ladder ReagentGeneRuler 100 bp DNA Ladder ready-to-useThermo Fisher ScientificCatalog #SM0243 . The eluate was then measured using a Fragment Analyzer with the High Sensitivity Kit.

Input DNA:

Ratio 1.8:

Ratio 1:

Ratio 0.9:

Ratio 0.85:

Ratio 0.8:

Ratio 0.75:

Ratio 0.7:

Ratio 0.65:

Ratio 0.6:

Ratio 0.55:



Materials
Materials required:
Below all materials needed for the protocol are listed. Vendors and part numbers are listed but interchangeable depending on the supply situation.

Chemicals:

Ethanol absolute ReagentEthanol absolute 99.8%Fisher ScientificCatalog #11994041
Hydrochloric acid fuming 37% ReagentHydrochloric acid fuming 37%Sigma AldrichCatalog #1003171011
Tris ultrapure 99.9%ReagentTris ultrapure 99.9%DiagonalCatalog #A1086.1000
EDTA disodium salt ReagentEDTA disodium saltSigma AldrichCatalog #E5134-50G
Tween 20 ReagentTween 20Carl RothCatalog #9127.1
Sera-Mag SpeedBeads ReagentSera-Mag SpeedBeads carboxylate modified particlesSigma AldrichCatalog #GE45152105050350

PCR-grade water ReagentInvitrogen UltraPure DNase/RNase-Free Distilled WaterFisher ScientificCatalog #11538646

Labware:

125 mL Nalgene Wide-Mouth Bottle ReagentThermo Scientific Nalgene Wide-Mouth LDPE Bottle with ClosureFisher ScientificCatalog #10044180
Large magnet ReagentNeodyme magnetMagnethandelCatalog #3935
96-well plate magnetReagentMM-Seperator M96Carl RothCatalog #2141.1
Hard-Shell PCR Plate ReagentHard-Shell 96-well PCR plateBioRad SciencesCatalog #HSP9601
Clear Polystyrene 96-Well Microplate ReagentCorning Clear Polystyrene 96-Well EIA/RIA MicroplateFisher ScientificCatalog #10380982

Stock solutions:

Amount1 L Tris stock solution Concentration1 Molarity (M) Ph8.5
  • Add Amount121.14 g Tris ultrapure 99.9% to a beaker
  • Adjust volume to Amount800 mL with ddH2O
  • Adjust pH to Ph8.5 with HCl
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L Tris stock solution Concentration1 Molarity (M) Ph8
  • Add Amount121.14 g Tris ultrapure 99.9% to a beaker
  • Adjust volume to Amount800 mL with ddH2O
  • Adjust pH to Ph8 with HCl
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L Tris stock solution Concentration1 Molarity (M) Ph7.5
  • Add Amount121.14 g Tris ultrapure 99.9% to a beaker
  • Adjust volume to Amount800 mL with ddH2O
  • Adjust pH to Ph7.5 with HCl
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L EDTA stock solution Concentration0.5 Molarity (M) Ph8
  • Add Amount186.12 g EDTA disodium salt to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Adjust pH to Ph8 with sodium hydroxide
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L wash buffer stock solution ( Concentration50 millimolar (mM) Tris )Ph7.5
  • Add Amount50 mL Tris stock solution Ph7.5 to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L PEG-NaCl buffer (Concentration2.5 Molarity (M) NaCl ,Concentration20 Mass / % volume PEG 8000 ,Concentration10 millimolar (mM) Tris ,Concentration1 millimolar (mM) EDTA ,Concentration0.05 % (v/v) Tween 20 )Ph8
  • Add Amount200 g PEG 8000 to a beaker
  • Add Amount146.2 g NaCl
  • Add Amount10 mL Tris stock solution Ph8
  • Add Amount2 mL EDTA stock solution Ph8
  • Add Amount250 µL of Tween 20
  • Adjust volume to Amount1 L with ddH2O
  • Dissolve the PEG and NaCl by stirring and heating to Temperature80 °C the solution will become milky at this point.
  • Let the solution cool down to TemperatureRoom temperature
  • Sterilize by filtering and store at Temperature4 °C

Working solutions:

Amount1 L TE minimum buffer (Concentration10 millimolar (mM) Tris , Concentration1 millimolar (mM) EDTA )Ph8
  • Add Amount10 mL Tris stock solution Ph8 to a beaker
  • Add Amount200 µL EDTA stock solution Ph8
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L wash buffer ( Concentration10 millimolar (mM) Tris , Concentration80 % (v/v) Ethanol )Ph7.5
  • Add Amount200 mL wash buffer stock solution
  • Adjust volume to Amount1 L with Ethanol absolute
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L elution buffer (Concentration10 millimolar (mM) Tris )Ph8.5
  • Add Amount10 mL Tris stock solution Ph8.5 to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount100 mL cleanup solution Ph8
  • Add Amount2 mL Sera-Mag SpeedBeads carboxylate modified to a clean Amount125 mL Nalgene bottle
  • Add Amount25 mL TE minimum buffer
  • Shake the bottle to wash the beads
  • Place the bottle on a large magnet for Duration00:05:00 to pellet the beads
  • Discard the supernatant
  • Add Amount25 mL TE minimum buffer
  • Shake the bottle to wash the beads
  • Place the bottle on a large magnet for Duration00:05:00 to pellet the beads
  • Discard the supernatant
  • Add Amount100 mL PEG-NaCl buffer
  • Shake well to resuspend the beads
  • Store at Temperature4 °C










Safety warnings
Reagents are potentially damaging to the environment. Dispose waste responsibly.
Before start
Make sure all buffers are prepared before starting.
For easier pipetting let the bead-solution adjust to TemperatureRoom temperature

Note
The protocol described here is designed for the use of Amount250 µL U-bottom assay plates 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.



Shake the cleanup solution until the beads are homogeneously resuspended

Note
The protocol described here uses a cleanup solution to sample ratio of 0.8:1. 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".

Add Amount30 µL PCR-grade water and Amount32 µL of cleanup solution to a Amount250 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 Amount2 µL the effect on the ratio is larger if working with a Amount10 µL assay than when working with a Amount80 µL assay.

The amount of beads is calculated as follows:
(sample volume + water volume) * ratio = cleanup solution volume

In this example:
(Amount10 µL PCR product +Amount30 µL PCR-grade water ) * 0.8 = Amount32 µL cleanup solution

For higher sample numbers PCR-grade water and cleanup solution can be prepared as a master mix.


Add Amount10 µL of sample.

Note
This protocol works for the cleanup of PCR products as well as the cleanup of DNA extracts or for buffer exchange after enzyme treatment of samples.

To bind the DNA to the beads shake at Shaker900 rpm, Room temperature , 00:05:00

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 Duration00:02:00

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

2m
Discard the supernatant by pipetting
With the plate still on the magnet, add Amount100 µL of wash buffer to each sample

Incubate for at least Duration00:00:30

30s
Discard the supernatant by pipetting
Go to and repeat once for a total of 2 washes

With the plate still on the magnet, incubate the plate for Duration00:05:00 atTemperatureRoom temperature to dry off residuals of wash buffer

5m
Add Amount40 µL of elution buffer to each sample

Shaker900 rpm, Room temperature , 00:05:00 to elute the DNA from the beads

Place the plate on a magnet to pellet the beads for Duration00:02:00
2m
Transfer Amount30 µL of the DNA to a new PCR plate. Store at Temperature-20 °C

Note
Leaving Amount10 µL 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.