TITLE: Guanidine-based DNA extraction with silica-coated beads or silica spin columns
AUTHORS: Dominik Buchner

[DESCRIPTION]
This protocol describes how to extract DNA from samples lysed as described in 
 
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. 

[BEFORE_START]
Make sure all buffers are prepared before starting.         

[GUIDELINES]
Follow general lab etiquette. Wear gloves to prevent contamination of samples. Clean the workspace before starting and after finishing with 80% EtOH.

[STEPS]
1. To clear the lysates 11.000 x g, 3 min, 20 °C 

SECTION: Bead-based protocol
2. Prepare 240 µL and 20 µL per sample in a 1.2 mL 
SECTION: Bead-based protocol
3. Add 100 µL 
 

SECTION: Bead-based protocol
4.     

SECTION: Bead-based protocol
5. Place the plate on a magnet to pellet the beads for 2 min 

 

SECTION: Bead-based protocol
6. Discard the supernatant by pipetting
SECTION: Bead-based protocol
7. Add 100 µL to each sample

SECTION: Bead-based protocol
8.   

SECTION: Bead-based protocol
9. Place the plate on a magnet to pellet the beads for 1 min 
SECTION: Bead-based protocol
10. Discard the supernatant by pipetting
SECTION: Bead-based protocol
11.   

SECTION: Bead-based protocol
12. Incubate the plate at 50 °C for 5 min 

SECTION: Bead-based protocol
13. Add 100 µL to each sample

SECTION: Bead-based protocol
14.   
 


SECTION: Bead-based protocol
15. Place the plate on a magnet to pellet the beads for 2 min 
SECTION: Bead-based protocol
16. Transfer 95 µL of the DNA to a new PCR plate. Store at -20 °C 

 


SECTION: Spin column protocol (centrifugation)
17. Combine 400 µL with 200 µL, vortex shortly

 

SECTION: Spin column protocol (centrifugation)
18. Load all of the volume on a silica spin column and 11.000 x g, 1 min  to bind the DNA, discard the flow-through

 



SECTION: Spin column protocol (centrifugation)
19. Add 600 µL to the spin column and 11.000 x g, 30 s , discard the flow-through

 


SECTION: Spin column protocol (centrifugation)
20.   and repeat for a total of 2 washes

SECTION: Spin column protocol (centrifugation)
21. 11.000 x g, 1 min to dry the silica membrane

SECTION: Spin column protocol (centrifugation)
22. Discard the collection tube and place the spin column in a clean 1.5 mL microcentrifuge tube

SECTION: Spin column protocol (centrifugation)
23. Add 100 µL directly to the silica membrane

SECTION: Spin column protocol (centrifugation)
24.  
Incubate for 3 min at Room temperature 

 


SECTION: Spin column protocol (centrifugation)
25. 11.000 x g, 1 min to elute the DNA. Discard the spin column, and store the eluted DNA at -20 °C 

SECTION: Spin column protocol (vacuum manifold)
26. Combine 400 µL with 200 µL, vortex shortly

 


SECTION: Spin column protocol (vacuum manifold)
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 (2 min ). Release the vacuum

 



SECTION: Spin column protocol (vacuum manifold)
28. Add 600 µL to the spin column or filter plate. Apply vacuum until all of the buffer has passed the column (1 min ). Release the vacuum

 


SECTION: Spin column protocol (vacuum manifold)
29.   and repeat for a total of 2 washes

SECTION: Spin column protocol (vacuum manifold)
30. Apply vacuum for 10 min to completely dry the silica membrane

 


SECTION: Spin column protocol (vacuum manifold)
30.1. For spin columns: 
  and follow the protocol for centrifugation

SECTION: Spin column protocol (vacuum manifold)
30.2. For 96-well filter plates:
Place a suitable collection plate in the vacuum manifold

 


SECTION: Spin column protocol (vacuum manifold)
30.3. Add 100 µL directly to the silica membrane. Apply vacuum until all of the elution buffer has passed the column ( 1 min ). Store eluted DNA at -20 °C