May 28, 2024
Sucrose lysis buffer
- Colleen Kellogg1
- 1Hakai Institute
- Pelagic Ecosystems Lab
- Hakai Genomics
Protocol Citation: Colleen Kellogg 2024. Sucrose lysis buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8op1l5r/v1
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 30, 2024
Protocol Integer ID: 100673
Keywords: eDNA, marine microbiology, biodiversity, environmental dna from seawater sample, dna on sterivex filter, seawater sample, plankton communities in the northern salish sea, preparation of sucrose lysis buffer, sucrose lysis buffer, part of the hakai institute ocean observing program, hakai institute ocean observing program, plankton community, environmental dna, sucrose lysi, biomolecular sample, sterivex filter, microbial, northern salish sea, prokaryote, prokaryotes to eukaryote, dna, eukaryote, pipeline, sequencing
Abstract
This protocol describes the preparation of sucrose lysis buffer to preserve DNA on sterivex filters. As part of the Hakai Institute Ocean Observing Program, biomolecular samples have been collected weekly, from 0 m to near bottom (260 m), to genetically characterize plankton communities in the Northern Salish Sea since 2015. This protocol is developed to work across all domains of life, from viruses to prokaryotes to eukaryotes, allowing for both amplicon sequencing and shotgun sequencing. The protocol is part of the Hakai Institute's pipeline to analyze microbial and environmental DNA from seawater samples and is implemented as a standard procedure for ongoing sampling programs.
Guidelines
MIOP: Minimum Information about an Omics Protocol
| MIOP Term | Value | |
| analyses | Nucleic Acid Water Filtration | |
| audience | scientists | |
| broad-scale environmental context | marine biome ENVO_00000447 | |
| creator | Colleen Kellogg | |
| environmental medium | sea water [ENVO:00002149] | |
| geographic location | North Pacific Ocean[GAZ:00002410] | |
| hasVersion | 1 | |
| issued | 2017 | |
| language | en | |
| license | CC BY 4.0 | |
| local environmental context | oceanic epipelagic zone biome [ENVO:01000033] | |
| materials required | Peristaltic Pump | |
| maturity level | Mature | |
| methodology category | Sample collection | |
| personnel required | 1 | |
| project | Hakai Institutes Marine Biodiversity | |
| publisher | Hakai Institute, Genomics Lab | |
| purpose | Sea water filtration [CHMO:0001640] | |
| skills required | sterile technique | pipetting skills | |
| target | DNA | |
| time required | 30 |
AUTHORS
| PREPARED BY | AFFILIATION | ORCID | DATE | |
| Colleen Kellogg | Hakai Institute | https://orcid.org/0000-0003-4048-5316 | 2017 |
RELATED PROTOCOLS
| PROTOCOL NAME AND LINK | ISSUER / AUTHOR | RELEASE / ACCESS DATE | |
| Suckrose Lysis Buffer | Hakai Institute |
This is a list of other protocols which should be known to users of this protocol. Please include the link to each related protocol.
ACRONYMS AND ABBREVIATIONS
| ACRONYM / ABBREVIATION | DEFINITION | |
GLOSSARY
| SPECIALISED TERM | DEFINITION | |
BACKGROUND
This document describes the required protocol to to filter seawater onto a 0.22 micrometer Sterivex filters using paristaltic pump setup.
Method description and rationale
This water filtration is part of the standard best - practice method for analysing microbial and environmental DNA from seawater samples at the Hakai Institutes Genome Lab. The method is part of a pipeline that includes seawater filtration, DNA extraction, and amplicon sequencing.
Spatial coverage and environments of relevance
As part of the Hakai Institute Ocean Observing Program, biomolecular samples have been collected weekly, from 0 to near bottom (260 m), to genetically characterize plankton communities in the Northern Salish Sea since 2015, developing a climatology from which we can begin uncover the physical, chemical and biological drivers of community and functional change in the dynamic coastal waters of coastal British Columbia. We work across all domains of life, from virus to prokaryotes to eukaryotes, employing both amplicon sequencing and shotgun sequencing.
Personnel Required
1 Technician
Safety
Identify hazards associated with the procedure and specify protective equipment and safety training required to safely execute the procedure!
Training requirements
Sterile technique, pipetting skills. Work-safe laboratory practices.
seawater
Materials
| DESCRIPTION e.g. filter | PRODUCT NAME AND MODEL Provide the official name of the product | MANUFACTURER Provide the name of the manufacturer of the product. | QUANTITY Provide quantities necessary for one application of the standard operating procedure (e.g. number of filters). | REMARK For example, some of the consumable may need to be sterilized, some commercial solution may need to be diluted or shielded from light during the operating procedure. | |
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Protocol materials
1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
UltraPure™ 0.5M EDTA, pH 8.0Thermo ScientificCatalog #15575020
sucroseFisher ScientificCatalog #BP220-1
Preparations
Note
Wear gloves and sterilize work area
You will need:
1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
UltraPure™ 0.5M EDTA, pH 8.0Thermo ScientificCatalog #15575020
sucroseFisher ScientificCatalog #BP220-1
- MilliQ Water
-500 mL bottle top filtration unit
Final concentrations of chemicals in SLB:
EDTA: 40 mM
Tris: 50 mM
Sucrose: 0.75 M
Calculations
Calculate how much Sucrose powder you need (using the molecular weight on the bottle, MW or FW) for a 0.75M solution of 500 mL.
Calculation of Sucrose:
Where Y is the molecular weight of the sucrose (MW or FW) from the bottle.
Calculation of Tris or EDTA:
Use the equation C1V1 = C2V2
Eg for EDTA:
(0.5M)(X mL) = (0.04M)(500 mL)
Solve for X
((0.04M)(500 mL)/(0.5M)) = 40 mL of 0.5M EDTA
Methods
Add the appropriate amount of Sucrose calculated above and place it in a clean bottle or beaker.
Add 40 mL of 0.5M EDTA to the beaker.
Add 25mL of 1M Tris to the beaker.
Add milliQ water to about the 400 mL line.
Add a stir bar and dissolve all the powder.
Top up water to 500 mL. (no need to pH this one!)
Filter-sterilize and label bottle.