Quick and dirty sequencing microbial genome extraction v1
Sebastian Cocioba, Sung won Lim
Abstract
Quick and dirty microbial genome extraction protocol using Edwards buffer.
I've been using this method for in-lab Archaeal evolution study for the last four years or so and it's been working fantastically well, especially for short read sequencing.
Certain microbes (such as Deinococcus) can be difficult to process properly using just this protocol - standard caveats and fixes tend to improve the output, such as adding initial lysozyme incubation step in the very beginning, sometimes combined with a freeze-thaw cycle for especially difficult samples (I'll update and upload a separate version of this protocol for difficult microbes in the future).
This particular protocol is aimed at getting as much intact, long-read capable DNA out from a microbe as cheaply as possible, as shown in the included gel picture.
Originally written up for my lab note at https://naturepoker.wordpress.com/2023/02/26/halobacteria-mutant-sequencing-3-years-in/
Ideation and initial testing performed with Sebastian S. Cocioba at Binomica Labs for ONT Deinococcus radiophilus genome sequencing project, using RAD004 rapid sequencing kit.
Steps
Spin down 1 ml of sample for 1 minute at max speed and decant
Resuspend vigorously with 100ul Edward's buffer
Transfer carefully to PCR tube - mixture will be viscous
Add 2ul of RNase A and mix vigorously, vortex for 10 seconds
Incubate at 37C for 15 minutes
Add 2ul of Proteinase K and mix vigorously, vortex for 10 seconds
Incubate at 55C for 1 hour, and deactivate via incubation at 95C for 10 minutes
Transfer to 1.5ml eppendorf tube
Add 10% 3M (pH 5.4) sodium acetate, and 1:1 volume of 100% isopropyl alcohol
Invert tube 10 times - precipitates should begin to form
Spin down at max speed for 5 minutes
Decant the supernatant carefully
Add 1ml of 70% EtOH and resuspend the pellet
Spin down at max speed for 5 minutes
Repeat 70% EtOH resuspension and washing step at least 2 more times
Decant completely and dry the pellet for 5 minutes - do not let the pellet overdry
Resuspend in storage buffer of choice or dH2O to wanted volume
Incubate in a 37C shaker for 1 hour - the extract is likely to be not dissolved fully
Incubate in 4C overnight - depending on yield, the extract will be extremely viscous
Check extraction quality using both standard gel electrophoresis and UV-Vis method such as Nanodrop.
