Sep 03, 2021
Version 2
FindingNemo Extraction 2: Phenol-free Method V.2
- 1University of Nottingham;
- 2University of British Columbia;
- 3University of Birmingham
Protocol Citation: Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose 2021. FindingNemo Extraction 2: Phenol-free Method. protocols.io https://dx.doi.org/10.17504/protocols.io.bxx2ppqeVersion created by Inswasti Cahyani
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 03, 2021
Last Modified: September 03, 2021
Protocol Integer ID: 52954
Keywords: ultra-long sequencing, cohex, glass bead, nanopore, MinION, UHMW DNA, Monarch, Circulomics, phenol, SDS, CTAB, GM12878, Whatman, PromethION, Nanobind
Abstract
This is a sub-protocol designed to extract/isolate ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers using a phenol-free extraction method.
A DNA extraction protocol that yields clean and homogeneous UHMW DNA is important for a good UL sequencing output. The choice of protocol should be based on achieving these parameters.
Kit-free, phenol-free protocol is a modification of NEB's Monarch HMW DNA Extraction Kit for Cells & Blood, with the option to use SDS or CTAB in the lysis buffer. This protocol also uses glass beads for DNA precipitation matrix.
We tested this sub-protocol in human cell line, with input cells of 3 millions but can be varied from 1-5 millions. As a rule of thumb, a million cells will suffice for one load on a MinION.
Guidelines
Acknowledgements
The FindingNemo protocol series was developed by Inswasti Cahyani for the Long Read Club with significant contributions from John Tyson and Nadine Holmes, also Josh Quick, and continuous discussion and support of Matt Loose and Nick Loman. We would also like to thank Giron Koetsier (NEB) and Kelvin Liu (Circulomics) for lending their expertise and advance product trials.
Please follow on Twitter for latest updates and results:
@NininUoN
@mattloose
Materials
Chemicals/Compounds
- 5M Ammonium AcetateSigma – AldrichCatalog #A-7330
- Tris-HCl pH 8.0Thermo ScientificCatalog #J22638-AE
- Ethanol AbsoluteHoneywellCatalog #32221-2.5L
- 1X Phosphate Buffer SalineFisher ScientificCatalog #15453819
- Proteinase K, 2mLQiagenCatalog #19131
- RNase AQiagenCatalog #19101
- Nuclease-free WaterThermofisherCatalog #AM9920
- NaCl (5 M) RNase-freeThermo Fisher ScientificCatalog #AM9759
- EDTA (0.5 M, pH 8.0, nuclease-free)Thermo Fisher ScientificCatalog #AM9260G
- 20% Sodium dodecyl sulfate (SDS)
- Cetyltrimethylammonium Bromide (CTAB)MP BiologicalsCatalog #02194004-CF
Made-up Buffer
Tris Lysis Buffer (TLB-SDS or TLB-CTAB)
- 100 mM NaCl
- 10 mM Tris-HCl, pH 8.0
- 25 mM EDTA, pH 8.0
- TLB-SDS = 0.5% (w/v) SDS or
- TLB-CTAB = 2% (w/v) CTAB
Disposables
- DNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
- DNA LoBind 2.0ml PCR Clean Eppendorf TubesEppendorfCatalog #0030 108.078
- Glass Beads 3 mmScientific Laboratory Supplies LtdCatalog #DD68501 OR Monarch DNA Capture BeadsNew England BiolabsCatalog #T3005L
- Thin-wall PCR Tubes 0.5 mlFisher ScientificCatalog #12194142
Note
cut tube 2-3 mm from the bottom to make a bead retainer
OR Monarch Bead RetainersNew England BiolabsCatalog #T3004L
- Monarch Collection Tubes II - 100 tubesNew England BiolabsCatalog #T2018L (optional)
Note
or use any 1.5 ml centrifuge tube as collection tube
- Wide-bore (or cut off) P1000 and P200 tips
Safety warnings
When handling phenol always wear PPE, keep a solution of 50% (w/v) PEG-400 nearby to treat the burn in the case of accidental splashes.
Before start
Things to observe at all times:
- Excessive and vigorous pipetting and vortexing should be avoided as these may shear the DNA.
- Make up buffers with nuclease-free water to avoid introducing nucleases to solutions.
- Avoid unnecessary heating and freezing; isolated DNA should be stable for storage in the fridge for months.
UHMW DNA Extraction
UHMW DNA Extraction
This protocol is adapted from Monarch® HMW DNA Extraction Kit for Cells & Blood.
Note
Either SDS (anionic surfactant) or CTAB (cationic surfactant) can be used in the lysis buffer.
Providing alternative surfactants in the lysis buffer may help with different cell systems that require different biochemistry.
Cell Lysis
Cell Lysis
5m
5m
Pellet 3 million cells in 1.5 ml tube by centrifuging at 1000 x g for 1 min at 4oC.
1000 x g, 4°C, 00:01:00
1m
Wash with PBS (make sure all media and serum are rinsed off), spin at 1000 x g for 1 min at 4oC.
1000 x g, 4°C, 00:01:00
1m
Add 149 μl of TLB-SDS or TLB-CTAB which has been added with 100 μg RNaseA (1 μl) and vortex at full speed for 3 seconds.
Note
Make master mix of the TLB and RNaseA if handling more than one sample.
Incubate at 37°C for 10 minutes.
37 °C 00:10:00
10m
Add 140 μl TLB and 200 μg Proteinase K (10 μl). Mix by slow inversion 5 times or with a P1000 wide-bore pipette tip.
Incubate at 55°C for 20 minutes.
55 °C 00:20:00
Note
Shaking in a thermomixer at 300-700 rpm can help lysis and homogenization, especially when more than 3 million cells are used.
20m
DNA Precipitation
DNA Precipitation
Add 75 μl of 5M Ammonium acetate, mix well with wide-bore P1000 pipette tip.
Add 3 clean glass beads to the cell lysate.
Add 275 μl of Isopropanol
Rotate the tube with a vertical rotator at 9 rpm for 5 minutes.
00:05:00 vertical rotator
Note
If a rotator is not available, hand inversion for 30-40 times can be used. Invert the tube slowly by hand so that a full inversion cycle takes 4-5 seconds.
5m
Remove and discard liquid by pipetting.
Wash bound DNA with 1 ml of 70% ethanol, invert tube 3 times, remove and discard ethanol.
Repeat step 13 once with 500 μl. Discard the ethanol, taking care not to disturb the DNA precipitate.
DNA Elution
DNA Elution
31m
31m
Insert a bead retainer to a collection tube.
Pour the beads into the bead retainer and spin for 1 s in a mini centrifuge (or the shortest time possible) to remove residual wash buffer. Keep the bead retainer.
Quickly pour the beads into a new 2 ml low-bind tube and immediately add 250 µl of elution buffer.
Note
Do not let the beads with DNA dry out. (As an alternative, 250 μl of elution buffer can be aliquoted into a 2 ml tube prior to this step.)
Incubate at 37oC for 30 min. Gently aspirate and dispense the eluate over the glass beads at regular intervals with a wide-bore P1000 tip to aid elution.
37 °C 00:30:00 mix per 10 min
30m
Insert the bead retainer from step 15 into a clean 2 ml DNA low-bind tube.
Pour the beads from step 17 and centrifuge at 12,000 x g for 1 minute.
12000 rpm, Room temperature, 00:01:00
1m
Quantify DNA as per "UHMW DNA QC" and check homogeneity by calculating %CV values. If the DNA is not sufficiently homegeneous, incubate the DNA for longer.
Store at 4oC or continue to UL Library Preparation as per "Modified ULK001".
If only SQK-RAD004 is available, follow library preparation in "Modified RAD004" or "KrazyStarFish (KSF)".
4 °C for storage