Oct 28, 2020
Ampure bead clean up for high molecular weight DNA
- 1The Ohio State University
- Sullivan Lab
Protocol Citation: Natalie Solonenko 2020. Ampure bead clean up for high molecular weight DNA . protocols.io https://dx.doi.org/10.17504/protocols.io.6kphcvn
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: August 16, 2019
Last Modified: October 28, 2020
Protocol Integer ID: 26991
Guidelines
For sample, do not vortex or mix by pipetting throughout protocol. This is to avoid shearing DNA.
Materials
MATERIALS
Agencourt AMPure XP beads
Before start
Turn on a heat block to 55 °C
Add beads
Add beads
Make sure beads are completely resuspended before use by vortexing vigorously.
Add ratio of resuspended beads specified in your main protocol.
Note
This ratio is dependent on the length of DNA you want to recover.
Flick tube gently to mix beads and sample.
Incubate
Incubate
Incubate at room temperature for 5 minutes.
Room temperature 00:05:00
Note
Flick gently periodically throughout the incubation to prevent beads from settling to the bottom of the tube.
Separation
Separation
Place tubes on a magnetic rack.
- see below for example.
Equipment
Magnetic Stand
NAME
Magnetic Stand
TYPE
Thermo Scientific
BRAND
MR02
SKU
LINK
Any magnetic rack that fits your tubes will suffice.
SPECIFICATIONS
Wait 2 min for the beads and buffer to seaparate. Beads will stick to magnetic side of the tube and the solution should be clear.
Remove and discard the clear solution. DNA will remain in the tube bound to the beads.
Wash
Wash
Add 500ul 80% EtOH to each sample.
- Do not disturb the beads! Pipette into the opposite side of the tube.
Note
Volume of 80% EtOH can be adjusted. Just so long as the amount added is enough to cover beads.
Incubate at room temperature for 30 seconds.
Room temperature 00:00:30
Remove 80% EtOH, being careful not to disturb the beads.
Repeat the step 4.
Spin down breifly and place back on the magnetic stand. Remove residual EtOH.
Dry Beads and Resuspend DNA
Dry Beads and Resuspend DNA
Leaving the caps open, remove tubes from magnet, and dry until surface of the beads has a matte finish.
Note
Caution: If the surface of beads appear cracked, they are overdrying! Resuspend immediately. Over-dried beads will not resuspend well.
Resuspend DNA by adding a minimun of 15ul water or low EDTA TE and gently mix by flicking. The exact resuspension volume should be specified in your main protocol.
Incubate for 2 minutes at 55 C.
55 °C 00:02:00
Collect
Collect
Place tubes back on magnetic rack
Wait 2 min for the beads to seaparate. Beads will stick to magnetic side of the tube and the solution should be clear.
Pipette out your specified volume of eluate and keep as your sample.
Note
Do not carry over any beads! They are a significant inhibitor of various downstream aplications. Carefully check to 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 ul.