Apr 15, 2025
Size-selection of sheared nematode DNA (~20 kb length) in a SPRI clean-up
- Manuela R. Kieninger1
- 1Blaxter Faculty, Wellcome Sanger, UK
Protocol Citation: Manuela R. Kieninger 2025. Size-selection of sheared nematode DNA (~20 kb length) in a SPRI clean-up. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkden1g5r/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 01, 2025
Last Modified: April 15, 2025
Protocol Integer ID: 125873
Keywords: DNA, HiFi sequencing, HMW DNA, SPRI, size selection, clean-up, genome, long read sequencing, reference genome, nematode, C. elegans
Funders Acknowledgements:
Wellcome Trust
Grant ID: 220540/Z/20/A
Abstract
This protocol describes the SPRI clean-up with size selection of sheared nematode DNA. After this SPRI clean-up the amount of low-molecular weight DNA (> 10 kb) will be significantly reduced. After QC the DNA can be used for low-input HiFi library preparation.
Guidelines
Make sure to pipette accurately for the sample and the beads.
Make sure to keep the supernatant after the initial bead incubation until you are sure, you got your DNA back.
Materials
- Magnetic rack for 1.5 and 2.0 ml tubes
- ProNex Size-Selective Chemistry (magnetic beads) (Promega) Cat. No. NG2003
- Wash Buffer (Promega) Cat. No. NG1051 (Component of ProNex‱ Size-Selective Purification System (Cat.# NG2001, NG2002, NG2003)
- Buffer EB (Qiagen) Cat. No. 19086
- 1000 µl, 200 µl, 10µl tips low-bind
- 200 µl bore tips low-bind
- Eppendorf DNA LoBind Tubes 1.5 ml Cat. No. 022431021
- Eppendorf DNA LoBind Tubes 2.0 ml Cat. No. 022431048
- Eppendorf‱ ThermoMixer‱ C Cat. No. 15158953
- Eppendorf Smart Block 2.0 ml Cat. No. 5362000035
- Mini centrifuge for 1.5 ml and 2.0 ml tubes
For QC:
- Qubit‱ 4 Fluorometer (Cat. No. Q33238)
- Qubit Assay tubes (Catalog number Q32856)
- Qubit reagents (for example Qubit 1X dsDNA HS Assay Kit Cat. No. Q33231)
- NanoDrop One (Cat. No. ND-ONE-W)
- Femto Pulse System (Agilent) (Cat. No. M5330AA)
- FemtoPulse reagents (Agilent): Genomic DNA 165 kb Kit (Cat. No. FP-1002-0275)
Safety warnings
- Please wear lab coat and nitrile gloves.
- Make sure to comply with the safety regulation in your laboratory.
- Please collect the waste in a suitable container and dispose of the waste in accordance with local regulations.
- On rare occasions it happens that the beads do not bind the DNA. I always keep the supernatant after the initial incubation step. If this happens the protocol can simply be repeated with the supernatant.
Before start
Make sure you added the required volume of ethanol to the Wash Buffer (Promega).
Take the ProNex beads (Promega) out of the fridge and warm them up to room temperature for 1 hr.
Make sure to know the exact volume of your DNA sample before you start the protocol.
Warm up the ThermoMixer to 37 degrees Celsius.
SPRI clean-up with ProNex beads
SPRI clean-up with ProNex beads
42m
42m
Put your DNA solution in a 2 mL LoBind tube. Make sure you know the volume exactly!
1m
Vortex the ProNex beads for 1 to 3 min.
(We have the beads aliquoted in 2 mL tubes for use.)
3m
Add 0.81x ProNex beads
It is important to be accurate! If DNA is limited you can use a 0.9x, but it will leave more low-molecular weight DNA behind.
1m
Mix well with bore tip (at least 20x) and incubate for 10 min at room temperature.
12m
Short spin the tube and put it on the magnetic rack.
30s
Leave it for 2 min.
2m
Take off supernatant.
Keep supernatant in case the beads did not bind the DNA!
30s
Add 2mL wash buffer and let it stand for 30 seconds. (The tube remains on the magnetic rack for the washing steps.)
30s
Take off the wash buffer and add again 2 mL wash buffer.
1m
Leave it for 30 seconds. Then take off the wash buffer.
30s
Close the tube and short spin the tube
30s
Put the tube back on the magnetic rack and take off the rest of the wash buffer.
30s
Wait 1 to 2 min (not longer) and add 50 µL Elution Buffer. Remove the tubes from the magnetic rack.
2m
Pipette mix with 200 µL bore tip thoroughly (at least 20x) and put the tube in the ThermoMixer at 37 degrees Celsius with 300 rpm for 15 min.
15m
Take the sample tubes back to the magnetic rack.
Wait 2 min until the liquid is completely clean and pipette DNA elution slowly to new 1.5 mL LoBind tube
It might be necessary to repeat this step if magnetic beads get taken over and are still in the solution. Make sure the solution is free from beads.
2m
Having a clean DNA solution, you can perform the QC:
Nanodrop, Qubit, FemtoPulse
QC:
- We measure the concentration using the Qubit.
- We measure concentration and purity of the DNA elution with the NanoDrop.
- We run the DNA on the FemtoPulse from Agilent to see the final fragment size distribution before library preparation.
If the QC is good, we proceed to low-input HiFi library preparation
Protocol references
Large DNA Size Selection with the ProNex Size-Selective Purification System.pdf