Oct 12, 2022
Version 2
Guanidine-based DNA extraction with silica-coated beads or silica spin columns V.2
- Dominik Buchner1
- 1University of Duisburg-Essen, Aquatic Ecosystem Research
- Aquatic Ecosystem Research
Protocol Citation: Dominik Buchner 2022. Guanidine-based DNA extraction with silica-coated beads or silica spin columns. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly73mmlx9/v2Version created by Dominik Buchner
Manuscript citation:
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: October 12, 2022
Last Modified: October 12, 2022
Protocol Integer ID: 71231
Keywords: dna extraction with silica, based dna extraction, dna from sample, silica spin column protocol, silica spin columns this protocol, silica spin column protocols higher yield, silica spin column, using guanidine hydrochloride, dna, guanidine hydrochloride, spin column protocol, guanidine, extraction, coated magnetic bead, approaches with magnetic bead, silica, magnetic bead, coated bead
Abstract
This protocol describes how to extract DNA from samples lysed as described in
Protocol
CREATED BY
Dominik Buchner
using guanidine hydrochloride and ethanol-based buffer combined with silica-coated magnetic beads or silica spin columns. The spin column protocol can be used either with centrifugation or, alternatively, a vacuum manifold. Compared to approaches with magnetic beads, with silica spin column protocols higher yields are possible since the amount of lysate used can be increased. The bead-based protocol is an automation-friendly alternative.
Guidelines
Follow general lab etiquette. Wear gloves to prevent contamination of samples. Clean the workspace before starting and after finishing with 80% EtOH.
Materials
Materials required:
Below all materials needed for the protocol are listed. Vendors and part numbers are listed but interchangeable depending on the supply situation.
Chemicals:
Guanidine hydrochloride Guanidine hydrochlorideFisher ScientificCatalog #10543325
Bis-Tris Bis-TrisCarl RothCatalog #9140.1
Ethanol absolute Ethanol absolute 99.8%Fisher ScientificCatalog #11994041
Phenol red indicator solutionPhenol red indicator solutionVWR International (Avantor)Catalog #HACH21132
Hydrochloric acid fuming 37% Hydrochloric acid fuming 37%Merck MilliporeSigma (Sigma-Aldrich)Catalog #1003171011
SeraSil-Mag 400 silica-coated beads SeraSil-Mag 400 silica coated superparamagnetic beadsMerck MilliporeSigma (Sigma-Aldrich)Catalog #GE29357371
Tris ultrapure 99.9%Tris ultrapure 99.9%DiagonalCatalog #A1086.1000
EDTA disodium salt EDTA disodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #E5134-50G
Sodium hydroxide Sodium hydroxide - pelletsFisher ScientificCatalog #S/4920/60
Labware:
50 mL Falcon tube Easy Reader Conical Polypropylene Centrifuge TubeFisher ScientificCatalog #11512303
125 mL Nalgene Wide-Mouth Bottle Thermo Scientific Nalgene Wide-Mouth LDPE Bottle with ClosureFisher ScientificCatalog #10044180
Large magnet Neodyme magnetMagnethandelCatalog #3935
1.2 mL square-well plate 1.2 mL square-well storage plateThermo Fisher ScientificCatalog #AB1127
96-well plate magnetMM-Seperator M96Carl RothCatalog #2141.1
Hard-Shell PCR Plate Hard-Shell 96-well PCR plateBio-Rad LaboratoriesCatalog #HSP9601
EconoSpin mini spin column EconoSpin mini spin clumn with lidEpoch Life ScienceCatalog #1920-050
1.5 mL Microcentrifuge tubes 1.5 mL microcentrifuge tubeSarstedtCatalog #72,690,001
EconoSpin 96-well filter plate EconoSpin 96-well filter plateEpoch Life ScienceCatalog #2020-001
Stock solutions:
50 mL Bis-Tris stock solution 1 Molarity (M)
- Add 10.5 g Bis-Tris to a 50 mL Falcon tube
- Adjust volume to 50 mL with ddH2O
- Vortex to completely dissolve the Bis-Tris
- Store at 4 °C
1 L Tris stock solution 1 Molarity (M) 8.5
- Add 121.14 g Tris ultrapure 99.9% to a beaker
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 8.5 with HCl
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L Tris stock solution 1 Molarity (M) 8
- Add 121.14 g Tris ultrapure 99.9% to a beaker
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 8 with HCl
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L Tris stock solution 1 Molarity (M) 7.5
- Add 121.14 g Tris ultrapure 99.9% to a beaker
- Adjust volume to 800 mL with ddH2O
- Adjust pH to 7.5 with HCl
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L EDTA stock solution 0.5 Molarity (M) 8
- Add 186.12 g EDTA disodium salt to a beaker
- Adjust volume to 1 L with ddH2O
- Adjust pH to 8 with sodium hydroxide
- Sterilize by filtering and store at Room temperature
1 L wash buffer stock solution ( 50 millimolar (mM) Tris )7.5
- Add 50 mL Tris stock solution 7.5 to a beaker
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
Working solutions:
1 L GuHCl binding buffer (3 Molarity (M) Guanidine hydrochloride ,10 millimolar (mM) Bis-Tris 90 % (v/v) Ethanol )6
- Add 286.6 g Guanidine hydrochloride in a beaker
- Adjust volume to 900 mL with Ethanol absolute
- Add 10 mL Bis-Tris stock solution
- Adjust volume to 980 mL with ddH2O
- Add 4 mL Phenol red indicator solution
- Dissolve the Guanidine hydrochloride by mixing on a magnetic stirrer
- Adjust to 6 with HCl
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
1 L TE minimum buffer 8
- Add 10 mL Tris stock solution 8 to a beaker
- Add 200 µL EDTA stock solution 8
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
100 mL silica beads working solution
- Add 5 mL SeraSil-Mag 400 beads to a clean 125 mL Nalgene bottle
- Add 25 mL TE minimum buffer
- Shake the bottle to wash the beads
- Place the bottle on a large magnet for 00:05:00 to pellett the beads
- Discard the supernatant
- Add 25 mL TE minimum buffer
- Shake the bottle to wash the beads
- Place the bottle on a large magnet for 00:05:00 to pellett the beads
- Discard the supernatant
- Add 100 mL TE minimum buffer
- Store at Room temperature
1 L wash buffer ( 10 millimolar (mM) Tris , 80 % (v/v) Ethanol )7.5
- Add 200 mL wash buffer stock solution
- Adjust volume to 1 L with Ethanol absolute
- Sterilize by filtering and store at Room temperature
1 L elution buffer (10 millimolar (mM) Tris )8.5
- Add 10 mL Tris stock solution 8.5 to a beaker
- Adjust volume to 1 L with ddH2O
- Sterilize by filtering and store at Room temperature
Troubleshooting
Safety warnings
Buffers containing guanidine produce highly reactive compounds when mixed with bleach. Don't mix the extraction waste with bleach or solutions that contain bleach.
Reagents are potentially damaging to the environment. Dispose waste as mandated.
Before start
Make sure all buffers are prepared before starting.
To clear the lysates 11.000 x g, 20°C, 00:03:00
3m
Bead-based protocol
2m
Prepare 240 µL GuHCl binding buffer and 20 µL silica beads working solution per sample in a 1.2 mL square well plate
Add 100 µL of the cleared lysate
Note
The amount of lysate used in this protocol is flexible as long as it fits the plate used in the protocol. If the amount is to be changed the amount of binding buffer has to be adjusted accoringly as well to maintain a constant ratio of lysate volume + 20 µL beads to binding buffer.
The binding buffer volume can be calculated as follows:
binding buffer volume = 2 x (lysate volume + 20 µL beads )
700 rpm, Room temperature , 00:05:00 to bind the DNA to the beads
Place the plate on a magnet to pellet the beads for 00:02:00
Note
Depending on the magnet and volume used separation times may vary and have to be adjusted accordingly.
2m
Discard the supernatant by pipetting
Add 100 µL of wash buffer to each sample
1000 rpm, Room temperature , 00:01:00 to wash excess salt off the beads
Place the plate on a magnet to pellet the beads for 00:01:00
1m
Discard the supernatant by pipetting
and repeat once for a total of 2 washes
Incubate the plate at 50 °C to dry off residuals of ethanol for 00:05:00
5m
Add 100 µL elution buffer to each sample
1000 rpm, Room temperature , 00:05:00 to elute the DNA from the beads
Note
Elution at 50 °C or with pre-warmed elution buffer may increase the yield.
Place the plate on a magnet to pellet the beads for 00:02:00
2m
Transfer 95 µL of the DNA to a new PCR plate. Store at -20 °C
Note
Leaving 5 µL of elution buffer is recommended to avoid carry-over of beads.
Spin column protocol (centrifugation)
1m
Combine 400 µL GuHCl binding buffer with 200 µL of the cleared lysate , vortex shortly
Note
The amount of lysate used in this protocol is flexible. The ratio of GuHCl binding buffer to lysate should remain 2:1.
Load all of the volume on a silica spin column and 11.000 x g, Room temperature, 00:01:00 to bind the DNA, discard the flow-through
Note
If the binding buffer - lysate mixture exceeds the bed volume of the spin column it has to be loaded as often as needed to pass the complete volume through the spin column.
1m
Add 600 µL of wash buffer to the spin column and 11.000 x g, Room temperature, 00:00:30 , discard the flow-through
Note
The amount of wash buffer should be adjusted to the maximum volume that has been loaded on the column to bind the DNA to remove all remaining traces of salts.
30s
and repeat for a total of 2 washes
11.000 x g, Room temperature, 00:01:00 to dry the silica membrane
1m
Discard the collection tube and place the spin column in a clean 1.5 mL microcentrifuge tube
Add 100 µL of elution buffer directly to the silica membrane
Incubate for 00:03:00 at Room temperature
Note
Yield might be increased by using elution buffer pre-warmed to 50 °C
3m
11.000 x g, Room temperature, 00:01:00 to elute the DNA. Discard the spin column, and store the eluted DNA at -20 °C
1m
Spin column protocol (vacuum manifold)
1m
Combine 400 µL GuHCl binding buffer with 200 µL of the cleared lysate , vortex shortly
Note
The amount of lysate used in this protocol is flexible. The ratio of GuHCl binding buffer to lysate should remain 2:1.
Load all of the volume on a silica spin column or 96-well filter plate placed in a vacuum manifold. Apply vacuum until all of the volume has passed the column (00:02:00 ). Release the vacuum
Note
If the binding buffer - lysate mixture exceeds the bed volume of the spin column or filter plate it has to be loaded as often as needed to pass the complete volume through the spin column or filter plate.
Times for application of vacuum may vary depending on the pump used. If a well clogs completely, carefully clean the membrane with a sterile pipette tip without piercing it.
2m
Add 600 µL of wash buffer to the spin column or filter plate. Apply vacuum until all of the buffer has passed the column (00:01:00 ). Release the vacuum
Note
The amount of wash buffer should be adjusted to the maximum volume that has been loaded on the column to bind the DNA to remove all remaining traces of salts.
1m
and repeat for a total of 2 washes
Apply vacuum for 00:10:00 to completely dry the silica membrane
Note
More time might be needed if a weaker pump is used. If traces of wash buffer remain on the membrane it should be dried at 50 °C for 00:05:00 on a heat block stacked inside of a 1.2 mL storage plate.
10m
For spin columns:
and follow the protocol for centrifugation
For 96-well filter plates:
Place a suitable collection plate in the vacuum manifold
Note
Depending on the elution volume different collection plates may be suitable. For large volumes a storage plate (1.2 mL or 2.2 mL) is recommended. For smaller volumes a 96-well PCR plate or a U-bottom assay plate is recommended.
Add 100 µL of elution buffer directly to the silica membrane. Apply vacuum until all of the elution buffer has passed the column ( 00:01:00 ). Store eluted DNA at -20 °C
Note
Yield might be increased by using elution buffer pre-warmed to 50 °C
1m