FindingNemo Extraction 2: Phenol-free Method
Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose
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.
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.
Steps
UHMW DNA Extraction
This protocol is adapted from Monarch® HMW DNA Extraction Kit for Cells & Blood.
Cell Lysis
Pellet 3 million cells in 1.5 ml tube by centrifuging at 1000 x g for 1 min at 4oC.
1000x g,4°C
Wash with PBS (make sure all media and serum are rinsed off), spin at 1000 x g for 1 min at 4oC.
1000x g,4°C
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.
Incubate at 37°C for 10 minutes.
37°C 0h 10m 0s
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 0h 20m 0s
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.
0h 5m 0s
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
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.
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 0h 30m 0s
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.
12000rpm
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
