Jan 29, 2020
Modified LSK109 ligation prep with needle shear and bead clean up
- John Tyson1
- 1Snutch Lab, UBC, Vancouver, BC, Canada
External link: https://www.longreadclub.org/mountain-protocol/
Protocol Citation: John Tyson 2020. Modified LSK109 ligation prep with needle shear and bead clean up. protocols.io https://dx.doi.org/10.17504/protocols.io.7emhjc6
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: September 17, 2019
Last Modified: January 29, 2020
Protocol Integer ID: 27821
Abstract
Modified LSK109 ligation prep using needle shear and bead purification
Using HMW DNA purified as above we have been performing 29G and 26G needle shears to produce fragmentation to around 18Kb and 34kb respectively (reported read N50’s after basecalling). Also by going into the library preparations with 5-10ug of DNA have been able to produce enough material to provide up to 10 library loads from a single prep. We have not been performing the FFPE repair during the end-prep step, and have been diluting the sample at the cleanup points in the protocol with EB to prevent bead clumping during 0.4-0.5x AMPureXP purifications when DNA fragments are large and at high concentrations. Shown below are the results from a batch of 12 runs we performed using this approach.
Materials
- HMW genomic DNA
- Buffer EB
- AMPureXP bead solution
- Qubit DNA HS kit
- 29G or 26G needle
- Ethanol
- Ultra II End-Prep Buffer (NEB)
- Ultra II End-Prep Enzyme mix (NEB)
- LNB Buffer
- AMX adapter
- Quick T4 DNA Ligase (NEB)
- LFB buffer
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
10 µg of HMW genomic DNA made up to 250 µL with EB and sheared with either a 29G or 26G needle using 20 passes.
- For 26G shear add 250 µL of EB (10mM Tris-Cl pH8.0) followed by250 µL AMPureXP bead solution.
- For 29G shear add 125 µL AMPureXP bead solution.
Note
After shearing is a good time to get a reliable DNA quantification using the Qubit DNA HS kit etc.
Mix by flicking.
Incubate for 00:10:00 at Room temperature followed by pelleting on a magnet.
Remove supernatant and keeping on the magnet wash 2x with 300 µL – 500 µL 80 % ethanol without disturbing the pellet.
Briefly spin, return to magnet and remove any residual 80 % ethanol with a pipette before allowing the bead pellet to air dry for ~00:02:00 .
Removed tube from the magnet
Resuspend the bead pellet in 51 µL EB by flicking.
Spin down.
Allow DNA to elute by incubation at 37 °C for 00:05:00 .
Place tube on magnet and remove eluted DNA sample to a fresh tube.
Quantify 1 µL of the DNA sample using the Qubit DNA HS kit to confirm DNA recovery (~6 µg ).
Then set up the following End-Prep reaction:
- 50 µL DNA sample
- 7 µL Ultra II End-Prep Buffer
- 3 µL Ultra II End-Prep Enzyme mix
Incubate at 20 °C for 00:30:00 followed by 65 °C for 00:30:00 .
Add 120 µL EB buffer followed by 72 µL AMPureXP bead solution.
Mix by flicking.
Incubate for 00:10:00 at Room temperature followed by pelleting on a magnet.
Remove supernatant and keeping on the magnet wash 2x with 300 µL 80 % ethanol without disturbing the pellet.
Briefly spin.
Return to magnet and remove any residual 80 % ethanol with a pipette before allowing the bead pellet to air dry for ~00:02:00 .
Remove tube from the magnet.
Resuspend the bead pellet in 67 µL EB by flicking.
Spin down and allow DNA to elute by incubation at 0 °C for 00:05:00 with occasional flicking.
Place tube on magnet and remove eluted DNA sample to a fresh tube.
Quantify 1 µL DNA sample using the Qubit DNA HS kit to confirm DNA recovery (~4 µg – 5 µg ).
Then set up the following Ligation reaction:
- 66 µL DNA sample
- 25 µL LNB buffer
- 5 µL AMX adapter
- 4 µL Quick T4 DNA Ligase
Incubate at 20 °C / Room temperature for 01:00:00 .
Add 100 µL EB buffer followed by 80 µL AMPureXP bead solution.
Mix by flicking.
Briefly spin.
Incubate for 00:10:00 at Room temperature followed by pelleting on a magnet.
Remove supernatant and keeping on the magnet wash 2x with250 µL of LFB buffer by removing from the magnet and flicking, briefly spin, and pelleting again on the magnet.
Remove any residual LFB buffer while on the magnet with a pipette after final pelleting.
Remove tube from the magnet.
Resuspend the bead pellet in 21 µL ONT-EB buffer by flicking.
Spin down and allow DNA to elute by incubation at 37 °C for 00:05:00 .
Place tube on magnet and pellet beads.
Remove eluted DNA sample to a fresh tube.
Quantify 1 µL DNA sample using the Qubit DNA HS kit to confirm DNA recovery (~2.5 µg ).
Library ready to be combined with SQB and LLB for loading onto flowcel (200 ng – 400 ng per load).