Feb 06, 2023
Bead clean-up (single tube)
- 1USDA
Protocol Citation: timothy.creed 2023. Bead clean-up (single tube). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldz5qxv5b/v1
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: April 14, 2022
Last Modified: November 07, 2023
Protocol Integer ID: 60811
Abstract
Protocol for purification of DNA using SPRI beads
Protocol materials
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Step 1
Prepare reagents
Prepare reagents
5m
5m
Prepare the following reagents/materials:
Fresh 70% Ethanol
Nuclease-free water or elution buffer
DNA sample/PCR product
Clean 1.5 mL tubes
Magnetic tube rack
Note
- Make sure beads are thoroughly mixed/vortexed to ensure they are well resuspended, the solution should be a homogenous brown color.
- Beads and sample must be brought to Room temperature before mixing.
Note
- Ethanol should be freshly prepared. Due to miscibility of EtOH and water, prepare by adding volumetric parts, not by bringing to volume.
5m
Add beads to sample
Add beads to sample
7m
7m
In a 1.5 mL tube, add resuspended beads to sample/PCR product in ratio specified in main protocol.
Note
The ratio is dependent on the length of DNA you want to recover.
1m
Flick tube gently to mix beads and sample.
Note
Pipette mixing may fragment the sample. Do not vortex to mix.
1m
Allow mixture to incubate for 00:05:00 at Room temperature
Note
Place tube on slow rotation mixer or flick gently periodically throughout the incubation to prevent beads from settling to the bottom of the tube.
5m
Separation
Separation
3m 30s
3m 30s
Place tube on a magnetic rack.
Equipment
Magnetic Stand
NAME
Magnetic Stand
TYPE
Thermo Scientific
BRAND
MR02
SKU
LINK
Any magnetic rack that fits your tubes will suffice.
SPECIFICATIONS
30s
Wait ~1-3 minutes for the beads and buffer to separate. Beads will stick to magnetic side of the tube and the solution should be clear.
2m
Using a pipettor, remove and discard the clear supernatant solution. DNA will remain in the tube bound to the beads.
1m
Ethanol Washing
Ethanol Washing
2m 30s
2m 30s
Add 500 μL 70% EtOH to tube
- Do not disturb the beads! Pipette into the opposite side of the tube.
Note
Volume of 70% EtOH can be adjusted. Just so long as the amount added is enough to cover beads.
30s
Incubate at Room temperature for 00:00:30
30s
Using a pipettor, remove and discard the EtOH. Be careful not to disturb or aspirate the beads.
30s
Repeat the Step 4 EtOH wash. Try to remove as much EtOH as possible without disturbing the beads.
Note
A quick spin-down may help to remove residual EtOH.
1m
Air dry beads
Air dry beads
1m
1m
Leaving tube cap open, allow beads to dry for a maximum of 90 seconds.
Note
-Do not over-dry beads. Over-dried beads do not resuspend well and can lead to a loss of DNA.
-If the surface of beads appear cracked, they are over-drying. Resuspend immediately.
1m
Resuspend
Resuspend
1m
1m
Remove tube from magnetic rack and resuspend DNA/beads by adding a minimum of 15 μL nuclease-free water or desired buffer.
Note
The exact resuspension volume should be specified in your main protocol. Use a smaller volume to increase concentration of DNA
30s
Flick tube gently to resuspend beads in elution.
30s
Incubate
Incubate
5m
5m
Incubate at Room temperature for 00:02:00 - 00:05:00
5m
Recovery
Recovery
2m
2m
Place tube back on magnetic rack. Allow beads and elution to separate for 00:01:00 .
1m
Slowly aspirate out clear supernatant, now containing DNA, and transfer to final clean container.
Note
Do not carry over any beads. They are a significant inhibitor of various downstream applications. Carefully check your pipette tip for bead carryover. If there are beads, replace the sample into the tube and wait another 2 min. You may need to decrease the volume removed from the tube by 1-2 μL.
1m