HMW gDNA extraction from prokaryotic cultures and cryo preservation stocks
Richard RKS Stöckl
long-read sequencing
nanopore sequencing
gDNA
DNA extraction
HMW gDNA
HMW DNA
prokaryotic cells
archaea
bacteria
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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 10mg/mL, in
incubate 0h 30m 0s at 37°C
add 5µL 20mg/mL) + 10µL 10mg/mL) and 15µL of 20Mass / % volume
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)
add 80µL of 10Mass / % volume 700millimolar (mM)
incubate at 65°C for 0h 30m 0s
Remove non-DNA components
add one volume (should be roughly 750µL) chloroform:isoamyl alcohol (24:1; 12.000rcf), transfer top (aqueous) phase to new tube
mix aqueous phase with equal volume phenol:chloroform:isoamyl alcohol (25:24:1; 12.000rcf) as before, and transfer top (aqueous) phase to new tube
mix aqueous phase with equal volume chloroform:isoamyl alcohol (24:1; 12.000rcf), and transfer top (aqueous) phase to new
Pellet and wash gDNA
add 0.6 volumes (roughly 360µL) of cold 100% 36µL) of 3Molarity (M) 5.2
incubate at -20°C overnight
centrifuge 16.000rcf , discard supernatant, air dry briefly
add 0.6volumes cold (-20°C) 70% (v/v) 16.000rcf , discard supernatant, air dry briefly
Optional: repeat once
resuspend in 50µL 4°C for several hours)
