Jan 13, 2025
Version 2
Bar-Seq Library Preparation and Pooling: Preparation of Sera-Mag SpeedBeads V.2
- 1NIST
Protocol Citation: Nina Alperovich, David Ross 2025. Bar-Seq Library Preparation and Pooling: Preparation of Sera-Mag SpeedBeads. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5r2jg47/v2Version created by Open Datasets Initiative
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: June 12, 2024
Last Modified: January 13, 2025
Protocol Integer ID: 115701
Keywords: Deep mutational scanning, protein sequence-function relationships, fitness landscape, laboratory automation
Disclaimer
The protocol outlined in this document was created as a part of Growth-based Quantitative Sequencing-GROQ-Seq) Platform. The GROQ-Seq platform was created under Align to Innovate’s Open Dataset Initiative. Align to Innovate is a non-profit research organization operating under open science principles with the goal of improving science research with programmable experiments. The Open Datasets Initiative is working to accelerate community-driven science with the use of automated labs to pioneer robust data collection methods and curated, high-fidelity, public biological datasets amenable to machine learning. This work was supported by Align to Innovate’s Open Datasets Initiative which receives philanthropic funding in part from Griffin Catalyst.
Abstract
This protocol describes the preparation of Sera-mag SpeedBeads with PEG-salt buffer for downstream use in the Bar-Seq Library Preparation and Pooling protocols (see the Automation Instructions & User Instructions).
Materials
Instruments:
- Magnetic separation rack compatible with 2 mL microcentrifuge tubes
- Non-magnetic tube rack
Reagents:
- 15 mL of Sera-mag SpeedBeads Solution (MilliporeSigma 65152105050250)
- 15 mL of TE Buffer, Composition: 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA (ThermoFisher 12090015)
- Polyethylene Glycol (PEG) 8000 (ThermoFisher 43443.36)
- Sodium Chloride (NaCl) 5M Solution (MilliporeSigma 59222C
- Tris-HCl 1M Solution, pH 8.0 (ThermoFisher J22638.K2)
- Ethylenediaminetetraacetic acid (EDTA) solution (Millipore Sigma E8008)
- Nuclease-free water (ThermoFisher 4387936)
- Tween 20 (MilliporeSigma P9416)
Consumables:
- 15 individual 2 mL microcentrifuge tubes (Celltreat 229446)
- 50 mL sterile conical tubes (ThermoFisher 339652)
Prepare SpeedBeeds in tubes
Prepare SpeedBeeds in tubes
Vortex 15 mL Sera-mag SpeedBeads solution until beads are uniformly distributed.
Transfer 1 mL to each of the fifteen 2 mL microcentrifuge tubes.
Place all 15 of the tubes on the tube magnetic separation rack until beads are drawn to magnet.
Remove the supernatant from each tube.
Perform the first wash step
Perform the first wash step
Add 1 mL TE buffer to each tube.
Remove the tubes from the magnetic stand and mix by vortexing
Return tubes to the magnetic separation rack and wait until beads are drawn to magnet.
Remove supernatant from each tube.
Perform the second wash step
Perform the second wash step
Add 1 mL TE buffer to each tube.
Remove the tubes from the magnetic stand and mix by vortexing
Return tubes to the magnetic separation rack and wait until beads are drawn to magnet.
Remove supernatant from each tube.
Resuspend the beads
Resuspend the beads
Add 1 mL TE to each tube and remove from magnet.
Fully resuspend by vortexing and set in a non-magnetic tube rack (not on magnetic stand).
- Note: Beads that have been washed and resuspended in TE can be stored in the refrigerator at this point and used for the following steps as needed.
SpeedBeads magnetic beads/PEG/NaCl buffer preparation
SpeedBeads magnetic beads/PEG/NaCl buffer preparation
Add 9 g PEG-8000 to a 50 mL sterile conical tube.
Add 10 mL 5 mol/L NaCl to the conical tube.
Add 500 µL 1 mol/L Tris-HCl, pH8 to the conical tube.
Add 500 µL 0.5 mol/L EDTA to the conical tube.
Fill the conical tube to ~45 mL using sterile nuclease free water.
Mix the conical tube by vortexing every 1-2 minutes until PEG goes into solution.
- When not vortexing, the tube can be placed on a heated shaker (e.g., Eppendorf ThermoMixer) at 50°C, 600 rpm, to reduce time required to dissolve the PEG
- When finished, the solution should be clear.
Add 27.5 μL Tween 20 to the conical tube and mix gently by inverting 4-6 times.
Mix two 1 mL aliquots of the SpeedBeads + TE solution until the solution is a uniform brown color, then transfer the solutions from both tubes to the 50 mL conical tube.
Fill the conical tube to the 50 mL mark with sterile nuclease free water
Gently mix until the solution is a uniformed brown color.
Wrap in the conical tube in foil (or place in dark container) and store at 4 °C until use.
Acknowledgements
This protocol was formatted for Protocols.io by Dana Cortade (Align to Innovate) and David Ross (NIST).