Oct 06, 2022

Public workspaceGuanidine-based DNA extraction with silica-coated beads or silica spin columns V.1

DOI
dx.doi.org/10.17504/protocols.io.eq2ly73mmlx9/v1
  • 1University of Duisburg-Essen, Aquatic Ecosystem Research
Dominik Buchner
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DOI: dx.doi.org/10.17504/protocols.io.eq2ly73mmlx9/v1
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/v1
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 05, 2022
Last Modified: October 06, 2022
Protocol Integer ID: 70847
Abstract
This protocol describes how to extract DNA from samples lysed as described in
Protocol
Sample preparation and lysis of homogenized malaise trap samples
NAME
Sample preparation and lysis of homogenized malaise trap samples
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 ReagentGuanidine hydrochlorideFisher ScientificCatalog #10543325
Bis-Tris ReagentBis-TrisCarl RothCatalog #9140.1
Ethanol absolute ReagentEthanol absolute 99.8%Fisher ScientificCatalog #11994041
Phenol red indicator solutionReagentPhenol red indicator solutionVWR international LtdCatalog #HACH21132
Hydrochloric acid fuming 37% ReagentHydrochloric acid fuming 37%Sigma AldrichCatalog #1003171011
SeraSil-Mag 400 silica-coated beads ReagentSeraSil-Mag 400 silica coated superparamagnetic beadsSigma AldrichCatalog #GE29357371
Tris ultrapure 99.9%ReagentTris ultrapure 99.9%DiagonalCatalog #A1086.1000
EDTA disodium salt ReagentEDTA disodium saltSigma AldrichCatalog #E5134-50G
Sodium hydroxide ReagentSodium hydroxide - pelletsFisher ScientificCatalog #S/4920/60


Labware:
50 mL Falcon tube ReagentEasy Reader Conical Polypropylene Centrifuge TubeFisher ScientificCatalog #11512303
125 mL Nalgene Wide-Mouth Bottle ReagentThermo Scientific Nalgene Wide-Mouth LDPE Bottle with ClosureFisher ScientificCatalog #10044180
Large magnet ReagentNeodyme magnetMagnethandelCatalog #3935
1.2 mL square-well plate Reagent1.2 mL square-well storage plateThermo Fisher ScientificCatalog #AB1127
96-well plate magnetReagentMM-Seperator M96Carl RothCatalog #2141.1
Hard-Shell PCR Plate ReagentHard-Shell 96-well PCR plateBioRad SciencesCatalog #HSP9601
EconoSpin mini spin column ReagentEconoSpin mini spin clumn with lidEpoch Life ScienceCatalog #1920-050
1.5 mL Microcentrifuge tubes Reagent1.5 mL microcentrifuge tubeSarstedtCatalog #72,690,001
EconoSpin 96-well filter plate ReagentEconoSpin 96-well filter plateEpoch Life ScienceCatalog #2020-001


Stock solutions:
Amount50 mL Bis-Tris stock solution Concentration1 Molarity (M)
  • Add Amount10.5 g Bis-Tris to a Amount50 mL Falcon tube
  • Adjust volume to Amount50 mL with ddH2O
  • Vortex to completely dissolve the Bis-Tris
  • Store at Temperature4 °C

Amount1 L Tris stock solution Concentration1 Molarity (M) Ph8.5
  • Add Amount121.14 g Tris ultrapure 99.9% to a beaker
  • Adjust volume to Amount800 mL with ddH2O
  • Adjust pH to Ph8.5 with HCl
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L Tris stock solution Concentration1 Molarity (M) Ph8
  • Add Amount121.14 g Tris ultrapure 99.9% to a beaker
  • Adjust volume to Amount800 mL with ddH2O
  • Adjust pH to Ph8 with HCl
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L Tris stock solution Concentration1 Molarity (M) Ph7.5
  • Add Amount121.14 g Tris ultrapure 99.9% to a beaker
  • Adjust volume to Amount800 mL with ddH2O
  • Adjust pH to Ph7.5 with HCl
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L EDTA stock solution Concentration0.5 Molarity (M) Ph8
  • Add Amount186.12 g EDTA disodium salt to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Adjust pH to Ph8 with sodium hydroxide
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L wash buffer stock solution ( Concentration50 millimolar (mM) Tris )Ph7.5
  • Add Amount50 mL Tris stock solution Ph7.5 to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Working solutions:

Amount1 L GuHCl binding buffer (Concentration3 Molarity (M) Guanidine hydrochloride ,Concentration10 millimolar (mM) Bis-Tris Concentration90 % (v/v) Ethanol )Ph6
  • Add Amount286.6 g Guanidine hydrochloride in a beaker
  • Adjust volume to Amount900 mL with Ethanol absolute
  • Add Amount10 mL Bis-Tris stock solution
  • Adjust volume to Amount980 mL with ddH2O
  • Add Amount4 mL Phenol red indicator solution
  • Dissolve the Guanidine hydrochloride by mixing on a magnetic stirrer
  • Adjust to Ph6 with HCl
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L TE minimum buffer Ph8
  • Add Amount10 mL Tris stock solution Ph8 to a beaker
  • Add Amount200 µL EDTA stock solution Ph8
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount100 mL silica beads working solution
  • Add Amount5 mL SeraSil-Mag 400 beads to a clean Amount125 mL Nalgene bottle
  • Add Amount25 mL TE minimum buffer
  • Shake the bottle to wash the beads
  • Place the bottle on a large magnet for Duration00:05:00 to pellett the beads
  • Discard the supernatant
  • Add Amount25 mL TE minimum buffer
  • Shake the bottle to wash the beads
  • Place the bottle on a large magnet for Duration00:05:00 to pellett the beads
  • Discard the supernatant
  • Add Amount100 mL TE minimum buffer
  • Store at TemperatureRoom temperature

Amount1 L wash buffer ( Concentration10 millimolar (mM) Tris , Concentration80 % (v/v) Ethanol )Ph7.5
  • Add Amount200 mL wash buffer stock solution
  • Adjust volume to Amount1 L with Ethanol absolute
  • Sterilize by filtering and store at TemperatureRoom temperature

Amount1 L elution buffer (Concentration10 millimolar (mM) Tris )Ph8.5
  • Add Amount10 mL Tris stock solution Ph8.5 to a beaker
  • Adjust volume to Amount1 L with ddH2O
  • Sterilize by filtering and store at TemperatureRoom temperature







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 Centrifigation11.000 x g, 20°C, 00:03:00

3m
Bead-based protocol
Bead-based protocol
2m
2m
Prepare Amount240 µL GuHCl binding buffer and Amount20 µL silica beads working solution per sample in a Amount1.2 mL square well plate
Add Amount100 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 + Amount20 µL beads to binding buffer.
The binding buffer volume can be calculated as follows:
binding buffer volume = 2 x (lysate volume + Amount20 µL beads )


Shaker700 rpm, Room temperature , 00:05:00 to bind the DNA to the beads

Place the plate on a magnet to pellet the beads for Duration00:02:00

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 Amount100 µL of wash buffer to each sample

Shaker1000 rpm, Room temperature , 00:01:00 to wash excess salt off the beads

Place the plate on a magnet to pellet the beads for Duration00:01:00
1m
Discard the supernatant by pipetting
Go togo to step #7 and repeat once for a total of 2 washes

Incubate the plate at Temperature50 °C to dry off residuals of ethanol

Add Amount100 µL elution buffer to each sample

Shaker1000 rpm, Room temperature , 00:05:00 to elute the DNA from the beads
Note
Elution at Temperature50 °C or with pre-warmed elution buffer may increase the yield.



Place the plate on a magnet to pellet the beads for Duration00:02:00
2m
Transfer Amount95 µL of the DNA to a new PCR plate. Store at Temperature-20 °C

Note
Leaving Amount5 µL of elution buffer is recommended to avoid carry-over of beads.



Spin column protocol (centrifugation)
Spin column protocol (centrifugation)
1m
1m
Combine Amount400 µL GuHCl binding buffer with Amount200 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 Centrifigation11.000 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 Amount600 µL of wash buffer to the spin column and Centrifigation11.000 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
Go togo to step #19 and repeat for a total of 2 washes

Centrifigation11.000 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 Amount1.5 mL microcentrifuge tube

Add Amount100 µL of elution buffer directly to the silica membrane

Incubate for Duration00:03:00 at TemperatureRoom temperature

Note
Yield might be increased by using elution buffer pre-warmed to Temperature50 °C



3m
Centrifigation11.000 x g, Room temperature, 00:01:00 to elute the DNA. Discard the spin column, and store the eluted DNA at Temperature-20 °C

1m
Spin column protocol (vacuum manifold)
Spin column protocol (vacuum manifold)
1m
1m
Combine Amount400 µL GuHCl binding buffer with Amount200 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 (Duration00:02:00 ). 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 Amount600 µL of wash buffer to the spin column or filter plate. Apply vacuum until all of the buffer has passed the column (Duration00:01:00 ). 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
Go togo to step #28 and repeat for a total of 2 washes

Apply vacuum for Duration00:10:00 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 Temperature50 °C for Duration00:05:00 on a heat block stacked inside of a 1.2 mL storage plate.



10m
For spin columns:
Go togo to step #23 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 Amount100 µL of elution buffer directly to the silica membrane. Apply vacuum until all of the elution buffer has passed the column ( Duration00:01:00 ). Store eluted DNA at Temperature-20 °C

Note
Yield might be increased by using elution buffer pre-warmed to Temperature50 °C


1m