Mar 07, 2023
AxyPrep magnetic Bead normalization and PCR clean up For dual index
- 1Natural History Museum of Utah
- Dentinger Lab
Protocol Citation: Alexander J Bradshaw 2023. AxyPrep magnetic Bead normalization and PCR clean up For dual index. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq4j13vk5/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: November 22, 2020
Last Modified: March 07, 2023
Protocol Integer ID: 44766
Keywords: pcr amplicons with axyprep bead, axyprep magnetic bead normalization, axyprep bead, pcr amplicon, axyprep, magnetic bead normalization, pcr, magnetic bead, bead
Abstract
In-house protocol used to prepare PCR amplicons with AxyPrep beads for Illumina sequencing.
Place Elution buffer, Magnetic Beads, and binding buffer on the bench and allow each to reach room temperature before using.
Room temperature
Mix magnetic beads until homogenous (no seriously, vortex the shit out of them)
Add 10ul of magnetic beads to each well. Pipette Up and down 5 times to mix completely
(10ul collects 200ng of DNA, adjust accordingly if more or less is desired)
add 100 ul of binding buffer (BB) to each well. Pipette Up and down 5 times to mix completely
Incubate samples at room temperature for 10 minutes
Room temperature 00:10:00
10m
place on magnetic separation device for 4 minutes or until the solution clears
00:04:00
4m
with the sample plate still, on the magnet, remove and discard the supernatant by pipetting.
Remove the sample plate from the magnetic separation device.
Add 150 μl freshly prepared 70% ethanol to each well
pipet mix 5 times and incubate for 2 minutes at room temperature.
00:02:00
2m
Place the sample plate on the 96 magnetic separation device for 4 minutes or until the solution clears.
00:04:00
4m
With the sample plate still on the magnet, remove and discard the supernatant by pipetting.
Dry the beads by incubating the plate for 2 minutes at room temperature with the plate still on the magnetic separation device.
.Remove the sample plate from the magnetic separation device.
00:02:00 Room temperature
2m
Add 50μl of EB-N Elution Buffer to each well and pipet up and down 5 times to mix.
Incubate the sample plate for 2 minutes at room temperature.
00:02:00 Room temperature
2m
Place the sample plate back on the magnetic separation device and wait 4 minutes or until the magnetic beads clear from the solution.
00:04:00
4m
Transfer the eluate (cleared supernatant) to a new plate/tube for storage or for subsequent applications.
For us, we will pool all 50ul of each sample into a single tube. If you have a lot of samples this could be a 50mL conical tube