May 28, 2024

Public workspaceSucrose lysis buffer

DOI
dx.doi.org/10.17504/protocols.io.j8nlk8op1l5r/v1
Sucrose lysis buffer
  • 1Hakai Institute
Andreas Novotny
Open access
DOI: dx.doi.org/10.17504/protocols.io.j8nlk8op1l5r/v1
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
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 TermValue
analysesNucleic Acid Water Filtration
audiencescientists
broad-scale environmental contextmarine biome ENVO_00000447
creatorColleen Kellogg
environmental mediumsea water [ENVO:00002149]
geographic locationNorth Pacific Ocean[GAZ:00002410]
hasVersion1
issued2017
languageen
licenseCC BY 4.0
local environmental contextoceanic epipelagic zone biome [ENVO:01000033]
materials requiredPeristaltic Pump
maturity levelMature
methodology categorySample collection
personnel required1
projectHakai Institutes Marine Biodiversity
publisherHakai Institute, Genomics Lab
purposeSea water filtration [CHMO:0001640]
skills requiredsterile technique | pipetting skills
targetDNA
time required30
AUTHORS
PREPARED BYAFFILIATIONORCIDDATE
Colleen KelloggHakai Institutehttps://orcid.org/0000-0003-4048-53162017

RELATED PROTOCOLS
PROTOCOL NAME AND LINKISSUER / AUTHORRELEASE / ACCESS DATE
Suckrose Lysis BufferHakai 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 / ABBREVIATIONDEFINITION
GLOSSARY
SPECIALISED TERMDEFINITION

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. filterPRODUCT NAME AND MODEL Provide the official name of the productMANUFACTURER 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.
Durable equipment
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Consumable equipment
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Chemicals
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Protocol materials
Reagent1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
Step 1
ReagentUltraPure™ 0.5M EDTA, pH 8.0Thermo ScientificCatalog #15575020
Step 1
ReagentsucroseFisher ScientificCatalog #BP220-1
Step 1
Preparations
Preparations

Note
Wear gloves and sterilize work area

You will need:
Reagent1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
ReagentUltraPure™ 0.5M EDTA, pH 8.0Thermo ScientificCatalog #15575020
ReagentsucroseFisher 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
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:
image.png
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
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.