Guanidine-based DNA extraction with silica-coated beads or silica spin columns

Dominik Buchner

Published: 2022-10-12 DOI: 10.17504/protocols.io.eq2ly73mmlx9/v2

Abstract

This protocol describes how to extract DNA from samples lysed as described in

Sample preparation and lysis of homogenized malaise trap samplesusing 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.

Before start

Make sure all buffers are prepared before starting.

Steps

To clear the lysates 11.000x g,20°C

Bead-based protocol

Prepare 240µL and 20µL per sample in a 1.2mL

Add 100µL

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 + 20µL to binding buffer. The binding buffer volume can be calculated as follows: binding buffer volume = 2 x (lysate volume + 20µL )

700rpm

Place the plate on a magnet to pellet the beads for 0h 2m 0s

Note

Depending on the magnet and volume used separation times may vary and have to be adjusted accordingly.

Discard the supernatant by pipetting

Add 100µL to each sample

1000rpm

Place the plate on a magnet to pellet the beads for 0h 1m 0s

10.

Discard the supernatant by pipetting

11.

12.

Incubate the plate at 50°C for 0h 5m 0s

13.

Add 100µL to each sample

14.

1000rpm

Note

Elution at 50°C or with pre-warmed elution buffer may increase the yield.

15.

Place the plate on a magnet to pellet the beads for 0h 2m 0s

16.

Transfer 95µL of the DNA to a new PCR plate. Store at -20°C

Note

Leaving 5µL of elution buffer is recommended to avoid carry-over of beads.

Spin column protocol (centrifugation)

17.

Combine 400µL with 200µL, 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.

18.

Load all of the volume on a silica spin column and 11.000x g 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.

19.

Add 600µL to the spin column and 11.000x g , 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.

20.

and repeat for a total of 2 washes

21.

11.000x g to dry the silica membrane

22.

Discard the collection tube and place the spin column in a clean 1.5mL microcentrifuge tube

23.

Add 100µL directly to the silica membrane

24.

Incubate for 0h 3m 0s at Room temperature

Note

Yield might be increased by using elution buffer pre-warmed to 50°C

25.

11.000x g to elute the DNA. Discard the spin column, and store the eluted DNA at -20°C

Spin column protocol (vacuum manifold)

26.

Combine 400µL with 200µL, 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.

27.

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 (0h 2m 0s ). 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.

28.

Add 600µL to the spin column or filter plate. Apply vacuum until all of the buffer has passed the column (0h 1m 0s ). 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.

29.

and repeat for a total of 2 washes

30.

Apply vacuum for 0h 10m 0s 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 0h 5m 0s on a heat block stacked inside of a 1.2 mL storage plate.

30.1.

For spin columns:

and follow the protocol for centrifugation

30.2.

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.

30.3.

Add 100µL directly to the silica membrane. Apply vacuum until all of the elution buffer has passed the column ( 0h 1m 0s ). Store eluted DNA at -20°C

Note

Yield might be increased by using elution buffer pre-warmed to 50°C