HMW gDNA extraction from prokaryotic cultures and cryo preservation stocks

Richard RKS Stöckl

Published: 2023-09-07 DOI: 10.17504/protocols.io.3byl4q7nrvo5/v1

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Abstract

This protocol can be used to extract High Molecular Weight gDNA from bacterial and archaeal cultures and cryo preservations thereof.

The resulting gDNA is usually suitable for long-read sequencing and is regularly used for genome assembly following Nanopore Sequencing.

It has been tested with a variety of mostly archaeal but also bacterial strains, including, but not limited to, Thermococcales , Thermotogales , E. coli , and Desulfurococcales .

Steps

Prepare cultures or cryopreservation capillaries

Transfer the bacteria-/archaea-suspension (~50µL) from one cryo preservation capillary to a 1.5 mL reaction tube or pellet a well-grown culture by centrifugation 15000rpm , discarding the supernatant, and resuspending the cell pellet in ~50µL media

Open up the cells

Add 490µL + 20µL freshly prepared solution (10mg/mL, in ) and vortex briefly

incubate 0h 30m 0s at 37°C

add 5µL (20mg/mL) + 10µL (10mg/mL) and 15µL of 20Mass / % volume , vortex briefly

incubate 1h 0m 0s at 56°C

freeze at -80°C for 0h 30m 0s and thaw at 60°C for 0h 10m 0s

repeat two additional times (total of three cycles)

add 100µL of 5Molarity (M) and mix well

Note

(this step is crucial as a CTAB–nucleic acid precipitate will form if salt concentration drops below about 0.5 M at room temperature)

add 80µL of 10Mass / % volume in 700millimolar (mM)

10.

incubate at 65°C for 0h 30m 0s

Remove non-DNA components

11.

add one volume (should be roughly 750µL) chloroform:isoamyl alcohol (24:1; ), shake vigorously, centrifuge full speed (> 12.000rcf), transfer top (aqueous) phase to new tube

12.

mix aqueous phase with equal volume phenol:chloroform:isoamyl alcohol (25:24:1; ), centrifuge full speed (>12.000rcf) as before, and transfer top (aqueous) phase to new tube

13.

mix aqueous phase with equal volume chloroform:isoamyl alcohol (24:1; ), centrifuge as before (>12.000rcf), and transfer top (aqueous) phase to new

Pellet and wash gDNA

14.

add 0.6 volumes (roughly 360µL) of cold 100% + 0.06 volumes (roughly 36µL) of 3Molarity (M) 5.2

15.

incubate at -20°C overnight

16.

centrifuge 16.000rcf , discard supernatant, air dry briefly

17.

add 0.6volumes cold (-20°C) 70% (v/v) , centrifuge at 16.000rcf , discard supernatant, air dry briefly

18.

Optional: repeat once

19.

resuspend in 50µL or or similar (ideally let this rest at 4°C for several hours)