May 31, 2024
Serapure Preparation and Testing
This protocol is a draft, published without a DOI.
- 1Hakai Institute
External link: http://hakai.org
Protocol Citation: rute.carvalho Carvalho 2024. Serapure Preparation and Testing. protocols.io https://protocols.io/view/serapure-preparation-and-testing-dejd3ci6
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: May 27, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 100677
Disclaimer
Draft!
Abstract
This protocol is used to prepare low-cost SPRI beads used for Illumina Library preparations. As part of the Hakai Institute Ocean Observing Program, from 0 m to near bottom (260 m), biomolecular samples have been collected weeklyto genetically characterize plankton communities in the Northern Salish Sea since 2015. These SPRI beads have been used to clean up PCR products of 16S, 18S, COI, and 12S amplicons, and implemented as part of a standard procedure for eDNA analysis.
This protocol is a modification from the following publication:
CITATION
Modified by B. Faircloth & T. Glenn November 19, 2011
Ecol. and Evol. Biology, UCLA
Protocol materials
NaCl (5 M), RNase-freeThermo FisherCatalog #AM9759
In 3 steps
GeneRuler 100 bp DNA Ladder Thermo Fisher ScientificCatalog #SM0241
Step 1
6-Tube Magnetic Separation RackNew England BiolabsCatalog #S1506S
Step 1
Sera-Mag SpeedBeads Carboxylate-Modified Magnetic ParticlesGE HealthcareCatalog #44152105050350
In 2 steps
PEG-8000 PromegaCatalog #V3011
In 2 steps
UltraPure™ 1M Tris-HCI pH 8.0Thermo Fisher ScientificCatalog #15568025
In 3 steps
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
In 3 steps
TWEEN 20 for molecular biology viscous liquidMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9416-50ML
In 2 steps
PREPARATIONS
PREPARATIONS
Materials:
Sera-Mag SpeedBeads Carboxylate-Modified Magnetic ParticlesGE HealthcareCatalog #44152105050350
PEG-8000 PromegaCatalog #V3011
UltraPure™ 1M Tris-HCI pH 8.0Thermo Fisher ScientificCatalog #15568025
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
TWEEN 20 for molecular biology viscous liquidMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9416-50ML
NaCl (5 M), RNase-freeThermo FisherCatalog #AM9759
GeneRuler 100 bp DNA Ladder Thermo Fisher ScientificCatalog #SM0241
6-Tube Magnetic Separation RackNew England BiolabsCatalog #S1506S
STEPS
STEPS
In a 50 mL conical using sterile stock solutions, prepare TE (10 mM Tris-HCl, 1 mM EDTA) by adding:
- 500 µL UltraPure™ 1M Tris-HCI pH 8.0Thermo Fisher ScientificCatalog #15568025
- 100 µL EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
- Fill conical to 50 mL mark with dH20.
Mix the container of Sera-Mag SpeedBeads Carboxylate-Modified Magnetic ParticlesGE HealthcareCatalog #44152105050350 and transfer 1 mL to a 1.5 mL microtube.
Place SpeedBeads on amagnet stand until beads are drawn to magnet.
Remove supernatant with P200 or P1000 pipetter.
Add 1 mL TE to beads, remove from the magnet, mix, and return to the magnet.
Remove supernatant with P200 or P1000 pipetter.
Add 1 mL TE to beads, remove from the magnet, mix, and return to the magnet.
Remove supernatant with P200 or P1000 pipetter.
Add 1 mL TE to beads and remove from magnet. Fully resuspend and set microtube in the rack (i.e. not on magnet stand).
Add 9 g PEG-8000 PromegaCatalog #V3011 to a new 50 mL, sterile conical.
Add 10 mL NaCl (5 M), RNase-freeThermo FisherCatalog #AM9759 (or 2.92 g NaCl) to conical.
Add UltraPure™ 1M Tris-HCI pH 8.0Thermo Fisher ScientificCatalog #15568025 to conical.
Add 100 uL EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G to conical.
Fill conical to ~ 49 mL using sterile dH20. You can do this by eye, just go slowly.
Mix conical for about 3-5 minutes until PEG goes into solution (solution, upon sitting, should be clear).
Add 27.5 µL TWEEN 20 for molecular biology viscous liquidMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9416-50ML to conical and mix gently.
Mix the 1mL SpeedBead + TE solution and transfer to 50 mL conical.
Fill conical to 50 mL mark with dH20 (if not already there) and gently mix 50 mL conical until brown.
Test against AMPure XP using aliquots of ladder (Fermentas GeneRuler). I recommend the 50 bp ladder in place of the ultraNlow range ladder.
Wrap in tinfoil (or place in dark container) and store at 4°C.
Note
You may also wish to prep an extra 50 mL of PEG solution that lacks Sera-mag SpeedBeads so that you can use it in a bead-inclusive library preparation protocol, derived from Fisher (2011).
In that case, just:
1. Add 10 g PEGN8000 to a new 50 mL, sterile conical.
2. Add 25 mL NaCl (5 M), RNase-freeThermo FisherCatalog #AM9759
(or 7.3 g NaCL) to conical.
3. Fill conical to ~ 49 mL using sterile dH20. You can do this by eye, just go slowly.
4. Mix conical for about 3-5 minutes until PEG goes into solution (solution, upon sitting,
should be clear).
Test monthly.
TESTING
TESTING
Note
You should test the Serapure mixture to ensure that it is working as expected. You can do this
using DNA ladder (Fermentas GeneRuler – NEB ladders may cause problems).
Prep fresh aliquots of 70% EtOH.
Mix 2 µL GeneRuler with 18 µL dH20.
Add 20 µL GeneRuler mixture to a volume of Serapure and/or AMPure (the specific volume depends on whether you are trying exclude small fragments or not; see the figure on the next page).
Incubate mixture for 5 min at room temperature.
Place on magnet stand.
Remove supernatant.
Add 500 µL 70% EtOH.
Incubate on stand for 1 min.
Remove supernatant.
Add 500 µL 70% EtOH.
Incubate on stand for 1 min.
Remove supernatant.
Place beads on 37°C heat block for 3-4 min. until dry.
Rehydrate with 20 µL dH20.
Place on magnet stand.
Transfer the supernatant to a new tube.
Mix supernatant with 1 µL loading dye.
Electrophorese in 1.5 % agarose for 1h at 100 V.
QUALITY CONTROL
QUALITY CONTROL
The following image compares the results of “purifying” a mix of 2 µL Fermentas Ultra Low
Range Ladder + 18 µL dH20 using several different amounts of AMPure or Serapure solution to
DNA solution. AMPure is on the left, “Serapure” is on the right. After preparing 20 µL of ladder
+ water mix, we combined that with the volumes of AMPure or Serapure listed below and then
purified using the standard protocol:
As you can see, the volume of AMPure or SeraPure controls the size of fragments recovered.
More specifically, the ratio of PEG solution used to the volume of the DNA in the solution
makes the difference, not the count of beads in the solution (provided they are above the minimum
level). This is what makes it possible to do “double-SPRI” size selection.
Citations
Rohland N, Reich D. . Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture.
https://doi.org/10.1101/gr.128124.111