Oct 17, 2016

Public workspaceFungal DNA Lysis Buffer

DOI
dx.doi.org/10.17504/protocols.io.fvfbn3n
  • Adam Taranto1
  • 1The Australian National University
  • Solomon Lab - Australian National University
Adam Taranto
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.fvfbn3n
Protocol Citation: Adam Taranto 2016. Fungal DNA Lysis Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.fvfbn3n
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 22, 2016
Last Modified: March 10, 2018
Protocol Integer ID: 3719
Abstract
Lysis buffer for extraction of DNA from fungal material. Contains ionic and non-ionic detergents. Contains RNAse-A.
Composition:
NaCl150mM
EDTA pH 8.01 mM
Tris Base pH 8.010 mM
Polyvinylpolypyrrolidone (PVPP)2%
IGEPAL-CA6300.5%
Sodium deoxycholate0.5%
SDS1%
RNAse-A100 µg/mL
Materials
MATERIALS
Reagent0.5mm diameter glass beadsScientific Industries, Inc.Catalog #SI-BG05
ReagentIGEPAL-CA630Merck MilliporeSigma (Sigma-Aldrich)Catalog #I3021 SIGMA-ALDRICH
ReagentTris(Hydroxymethyl)aminomethaneCatalog #PubChem CID: 6503
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentSodium DeoxycholateCatalog #PubChem CID: 23668196
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531
ReagentPolyvinylpolypyrrolidoneCatalog #CID 6917
STEP MATERIALS
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
ReagentTris HydroxymethylaminomethaneCatalog #PubChem CID: 6503
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentIGEPAL-CA630Merck MilliporeSigma (Sigma-Aldrich)Catalog #I3021 SIGMA-ALDRICH
ReagentSodium DeoxycholateCatalog #PubChem CID: 23668196
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
ReagentTris HydroxymethylaminomethaneCatalog #PubChem CID: 6503
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531
ReagentPolyvinylpolypyrrolidoneCatalog #CID 6917
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
ReagentTris HydroxymethylaminomethaneCatalog #PubChem CID: 6503
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentIGEPAL-CA630Merck MilliporeSigma (Sigma-Aldrich)Catalog #I3021 SIGMA-ALDRICH
ReagentSodium DeoxycholateCatalog #PubChem CID: 23668196
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
ReagentTris HydroxymethylaminomethaneCatalog #PubChem CID: 6503
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531
ReagentPolyvinylpolypyrrolidoneCatalog #CID 6917
Protocol materials
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531
ReagentPolyvinylpolypyrrolidoneCatalog #CID 6917
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
ReagentTris HydroxymethylaminomethaneCatalog #PubChem CID: 6503
ReagentSodium DeoxycholateCatalog #PubChem CID: 23668196
ReagentSodium DeoxycholateCatalog #PubChem CID: 23668196
ReagentIGEPAL-CA630Merck MilliporeSigma (Sigma-Aldrich)Catalog #I3021 SIGMA-ALDRICH
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
ReagentTris HydroxymethylaminomethaneCatalog #PubChem CID: 6503
ReagentSodium DeoxycholateCatalog #PubChem CID: 23668196
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
ReagentPolyvinylpolypyrrolidoneCatalog #CID 6917
ReagentTris HydroxymethylaminomethaneCatalog #PubChem CID: 6503
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
Reagent0.5mm diameter glass beadsScientific Industries, Inc.Catalog #SI-BG05
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
ReagentIGEPAL-CA630Merck MilliporeSigma (Sigma-Aldrich)Catalog #I3021 SIGMA-ALDRICH
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
ReagentPolyvinylpolypyrrolidoneCatalog #CID 6917
ReagentTris HydroxymethylaminomethaneCatalog #PubChem CID: 6503
ReagentIGEPAL-CA630Merck MilliporeSigma (Sigma-Aldrich)Catalog #I3021 SIGMA-ALDRICH
ReagentTris(Hydroxymethyl)aminomethaneCatalog #PubChem CID: 6503
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
ReagentTris HydroxymethylaminomethaneCatalog #PubChem CID: 6503
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentIGEPAL-CA630Merck MilliporeSigma (Sigma-Aldrich)Catalog #I3021 SIGMA-ALDRICH
ReagentSodium DeoxycholateCatalog #PubChem CID: 23668196
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
ReagentTris HydroxymethylaminomethaneCatalog #PubChem CID: 6503
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531
ReagentPolyvinylpolypyrrolidoneCatalog #CID 6917
Prepare Stock Solutions
Prepare Stock Solutions
10% SDS solution
Dissolve 10 g SDS in 80 mL ddH2O
Adjust volume to 100 mL
Amount10 g
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
1M Tris Base, pH 8.0
Dissolve 12.114g Tris-Base in 80 mL ddH2O
pH to 8.0 with 10 M HCl
Adjust volume to 100mL
Amount12 g
ReagentTris HydroxymethylaminomethaneCatalog #PubChem CID: 6503
0.5 M EDTA, pH 8.0
Add 18.61 g of disodium EDTA•2H2O to 80 mL of ddH2O
Adjust the pH to 8.0 with NaOH (~20 NaOH pellets)
Adjust volume to 100 mL
Amount19 g
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
3 M NaCl 
Dissolve 17.532 g NaCl in 80 mL ddH2O
Adjust volume to 100 mL
Amount18 g
ReagentSodium ChlorideCatalog #PubChem CID: 5234
Prepare Lysis Solution - 100 mL
Prepare Lysis Solution - 100 mL
Add 500µL IGEPAL-CA630 to 82.3 mL ddH2O
Stir until dissolved
Amount500 µL
Note
Final concentration: 0.5%
ReagentIGEPAL-CA630Merck MilliporeSigma (Sigma-Aldrich)Catalog #I3021 SIGMA-ALDRICH
Add 0.5g Sodium deoxycholate
Stir until dissolved
Note
Final concentration: 0.5%
ReagentSodium DeoxycholateCatalog #PubChem CID: 23668196
Add PVPP
Add 2g Polyvinylpolypyrrolidone, stir until dissolved.
Amount2 g
Note
Final concentration 2%
Note
PVPP precipitates phenols, tannins, alkaloids which may inhibit enzymes or damage DNA
ReagentPolyvinylpolypyrrolidoneCatalog #CID 6917
Add 10 mL of 10% SDS stock solution
Stir until dissolved
Amount10 mL
Note
Final concentration: 1%
Note
1% SDS = 34.67 mM
ReagentSodium Dodecyl SulfateCatalog #PubChem CID: 3423265
Add 5 mL of 3M NaCl stock solution
Amount5 mL
Note
Final concentration: 150 mM
ReagentSodium ChlorideCatalog #PubChem CID: 5234
Add 200 µL of 0.5 M EDTA (pH 8.0) stock solution
Amount200 µL
Note
Final concentration: 1 mM
ReagentEDTA Disodium SaltCatalog #PubChem CID: 8759
Add 1 mL Tris-Base (pH 8.0)
Stir until dissolved
Amount1 mL
Note
Final concentration: 10 mM
ReagentTris HydroxymethylaminomethaneCatalog #PubChem CID: 6503
Add 1mL of RNase A (10 mg/mL) stock solution
Amount1 mL
Note
Final concentration: 100 µg/mL
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531
Store lysis solution at 4˚C