Dec 02, 2021

Public workspaceTissue Culture (tc) PBS

DOI
dx.doi.org/10.17504/protocols.io.byajpscn
  • 1FDA
  • GenomeTrakr
    Tech. support email: genomeTrakr@fda.hhs.gov
Jessica L Jones
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DOI: dx.doi.org/10.17504/protocols.io.byajpscn
Protocol Citation: Jacquelina.Woods 2021. Tissue Culture (tc) PBS. protocols.io https://dx.doi.org/10.17504/protocols.io.byajpscn
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
Created: September 16, 2021
Last Modified: December 02, 2021
Protocol Integer ID: 53291
Abstract
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method provides a reagent formula required in the rapid concentration of intact viruses from wastewater using a combination of PEG precipitation and ultracentrifugation.
Materials
Equipment:
Scientific Accumet XL 500 pH meter (Fisher Scientific Cat # 16-636-XL500A, or similar)

Reagents:
ReagentSodium Chloride (NaCl)Sigma AldrichCatalog #S3014 , or equivalent
ReagentPotassium ChlorideSigma AldrichCatalog #P9541 , or equivalent
ReagentPotassium Phosphate dibasic (KH2PO4)Sigma AldrichCatalog #P9791 , or equivalent
ReagentSodium Phosphate dibasic anhydrous (Na2HPO4)Sigma AldrichCatalog #S5011 , or equivalent
Protocol materials
ReagentSodium Chloride (NaCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S3014
ReagentPotassium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9541
ReagentPotassium Phosphate dibasic (KH2PO4)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9791
ReagentSodium Phosphate dibasic anhydrous (Na2HPO4)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5011
ReagentSodium Chloride (NaCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S3014
ReagentPotassium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9541
ReagentPotassium Phosphate dibasic (KH2PO4)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9791
ReagentSodium Phosphate dibasic anhydrous (Na2HPO4)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5011
1X PBS
1X PBS
Dissolve components in deionized or ultrapure water to 1L.
Amount8.0 g ReagentSodium Chloride (NaCl)Sigma AldrichCatalog #S3014

Amount0.2 g ReagentPotassium ChlorideSigma AldrichCatalog #P9541

Amount0.12 g ReagentPotassium Phosphate dibasic (KH2PO4)Sigma AldrichCatalog #P9791

Amount0.91 g ReagentSodium Phosphate dibasic anhydrous (Na2HPO4)Sigma AldrichCatalog #S5011

Adjust Ph7.5

Autoclave Temperature121 °C Duration00:15:00

15m
Store at Temperature2-8 °C

Note
For 2X tc PBS, double components in 1.1 - 1.4


10X tc PBS
10X tc PBS

Note
Alternatively, 1X and 2X PBS can be made by dilution of Reagent10X PBSSigma AldrichCatalog #P5493 into sterile deionized or ultrapure water.