FindingNemo Extraction 3: NEB Monarch Kit

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 New England Biolabs (NEB) Monarch HMW DNA Extraction Kit for Cells & Blood .

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.

The New England Biolabs (NEB) Monarch HMW DNA Extraction Kit for Cells & Blood is a quick, tweakable extraction protocol fitting for one-day library prep.

We tested this sub-protocol in human cell line , with input cells varied between 1-6 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 using Monarch® HMW DNA Extraction Kit for Cells & Blood .

We have obtained optimal extractions using the NEB Monarch kit. This combines speed with high quality UHMW DNA. Follow the manufacturer’s instructions as described here, BUT incorporate the following changes as described below.

Note

Our most homogeneous extracted DNA samples were obtained by lysis at 600-700 rpm speed using the Monarch kit. This is one area that can be optimised depending on the input sample.

One-day Protocol

To complete DNA extraction from cell to library prep and sequencing in one day, start early!

Note

One million human cells are sufficient for a single library load on the MinION. At least two million human cells are required for a single load on the PromethION. Other samples can be scaled according to total amount of DNA recovered. So for a 1 gigabase genome you would require 3 million cells etc.

Dilute the eluted DNA with 150 μl of NEB Elution Buffer II.

Note

This is following the NEB UHMW Monarch kit protocol where DNA is first eluted with 100 μl buffer. After this step, sample volume will be 250 μl total. Quantification of a very viscous UHMW DNA is problematic and will not produce accurate results, hence the dilution. Gradual dilution is recommended to achieve homogeneous concentration of 50-100 ng/μl.

Incubate the eluted DNA at 37^oC for about 2-3 hours with regular pipette mixing.

37°C 3h 0m 0s

Note

During mixing, observe by eye that the viscous DNA 'blob' has been more or less dissolved to the different parts of the tube (i.e. , less heterogeneous). This is usually observed after 2 hours of incubation. Otherwise, continue the incubation to 3 hours and proceed to the next step.

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 4^oC or continue to UL Library Preparation as per " Modified ULK001 ".

If only SQK-RAD004 is available, follow library preparation as in " Modified RAD004 " or " KrazyStarFish (KSF) ".

4°C

Abstract

Before start

Steps

UHMW DNA Extraction

One-day Protocol

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