Serapure Beads Preparation and Testing
Colleen Kellogg, Matt Lemay, rute.carvalho Carvalho
Disclaimer
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 weekly to 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 protocol:
B. Faircloth and T. Glenn. Serapure v2.2. Ecol. and Evol. Biology, UCLA, November 19, 2011 Serapure v2.2. Ecol. and Evol. Biology, UCLA, November 19, 2011
Which was based on the following article:
Before start
Steps
PREPARATIONS
Materials (or equivalent):
STEPS
In a 50 mL conical using sterile stock solutions, prepare TE (10 mM Tris-HCl, 1 mM EDTA) by adding :
- 500 µL
- 100 µL
- Fill conical to 50 mL mark with dH20.
Mix the container of
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
Add 10 mL
Add
Add 100 uL
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
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.
Test monthly.
TESTING
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.
