Oct 19, 2022

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

DOI
dx.doi.org/10.17504/protocols.io.q26g7y859gwz/v3
  • 1University of Duisburg-Essen, Aquatic Ecosystem Research
Dominik Buchner
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DOI: dx.doi.org/10.17504/protocols.io.q26g7y859gwz/v3
Protocol Citation: Dominik Buchner 2022. PCR normalization and size selection with magnetic beads. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7y859gwz/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: 71542
Keywords: pcr cleanup, normalization, magnetic beads, library prep
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.
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 LadderReagentGeneRuler 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 ThermReagentThermo Scientific Nalgene Wide-Mouth LDPE Bottle with ClosureFisher ScientificCatalog #10044180
Large magnet ReagentNeodyme magnetMagnethandelCatalog #3935
96-well plate magnet ReagentMM-Seperator M96Carl RothCatalog #2141.1
Hard-Shell PCR Plate Hard-Shell 96-wellReagentHard-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

Amount100 mL normalization solution Ph8
  • Add Amount95 mL PEG-NaCl buffer to a clean Amount125 mL Nalgene bottle
  • Add Amount5 mL cleanup solution
  • 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 normalization solution adjust to Room 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 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 Amount9 µL PCR product . 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 Concentration2 Mass Percent .


Add Amount31 µL PCR-grade water and Amount28 µL of normalization solution 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 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:
(9 µL PCR product +31 µL PCR-grade water ) * 0.7 = 28 µL cleanup solution

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



Add Amount9 µL of PCR product

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
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.


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 at TemperatureRoom temperature to dry off residuals of wash buffer

5m
Add Amount50 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

Note
The bead pellet might be barely visible at this point.


2m
Transfer Amount40 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.