TITLE: Sucrose lysis buffer
AUTHORS: Colleen Kellogg

[DESCRIPTION]
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
 
 
 
 
AUTHORS
 

RELATED PROTOCOLS
 
 
ACRONYMS AND ABBREVIATIONS
 
 
GLOSSARY
 
 

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

[STEPS]
SECTION: Methods
10. Filter-sterilize and label bottle.
SECTION: Methods
9. Top up water to 500 mL. (no need to pH this one!)
SECTION: Methods
8. Add a stir bar and dissolve all the powder.
SECTION: Methods
7. Add milliQ water to about the 400 mL line.
SECTION: Methods
6. Add 25mL of 1M Tris to the beaker.
SECTION: Methods
5. Add 40 mL of 0.5M EDTA to the beaker.
SECTION: Methods
4. Add the appropriate amount of Sucrose calculated above and place it in a clean bottle or beaker.
SECTION: Calculations
2. 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:
 

SECTION: Preparations
1. 
 

You will need: 
  
  
  
- MilliQ Water
-500 mL bottle top filtration unit 

Final concentrations of chemicals in SLB: 
EDTA: 40 mM 
Tris: 50 mM 
Sucrose: 0.75 M 
SECTION: Calculations
3. 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