Oct 24, 2020
Version 2
DNA-Extraction-ClostridiaProject V.2
This protocol is a draft, published without a DOI.
- 1Universität Leipzig
Protocol Citation: Tobias Wandt 2020. DNA-Extraction-ClostridiaProject. protocols.io https://protocols.io/view/dna-extraction-clostridiaproject-bnw3mfgnVersion created by Tobias Wandt
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
I use this protocol and it works for me
Created: October 24, 2020
Last Modified: October 24, 2020
Protocol Integer ID: 43707
Abstract
Protocol used for DNA-Extraction of Clostridia in my project.
DNA-Kit used is GenFind V3 by Beckmann/Coulter.
Some steps used from:
Sim JH, Anikst V, Lohith A, Pourmand N, Banaei N. Optimized Protocol for Simple Extraction of High-Quality Genomic DNA from Clostridium difficile for Whole-Genome Sequencing. J Clin Microbiol. 2015;53(7):2329-2331. doi:10.1128/JCM.00956-15
Growing and Preparing Clostridia
Growing and Preparing Clostridia
2d
2d
Inoculate Clostridia in 10ml TSB-Broth, incubating at least 48h in anaerobe atmosphere at 37°C
10 mL TSB broth 48:00:00 at 37°C anaerobic
2d
Take 2ml of Clostridia TSB-Broth and centrifuge for 15min at 3,000g, after that remove supernatant
2 mL Clostridia TSB broth 00:15:00 centrifuge at 3000g
15m
DNA Extraction
DNA Extraction
1h 32m
1h 32m
Add 500ul Lysis-Buffer(LBB) and 30ul Proteinase K, tipmixing or vortexing to resuspend the pellet
500 µL LBB 30 µL Proteinase K
Incubate at 57°C for 1h while continously shaking at ~500/min
01:00:00 at 57°C + shaking 500/min
1h
Add 300ul vortexed Black Beads(BBB) and tipmix(at least 10 times), incubate for 5 min at room temperature, place on a magnetic tube stand until no or few beads are left in suspension ~10-15min, then carefully remove supernatant without disturbing the beads-formation
300 µL Black Beads 00:05:00 at room temperature 00:15:00 magnetic tube stand
20m
Add 800ul WBB and tipmix(at least 10 times), place on a magnetic tube stand until no or few beads are left in suspension ~5min, remove supernatant
800 µL WBB 00:05:00 magnetic tube stand
5m
go to step #6 for a total of 2 WBB washes
Add 800ul WBC and tipmix (at least 10 times), than add 600ul WBC and tipmix, place on a magnetic tube stand until no or few beads left in suspension ~5min , remove supernatant
1400 µL WBC 00:05:00 magnetic tube stand
5m
go to step #8 for a total of 2 WBC washes
Add 100ul distilled water, tipmix 10 times, incubate for 2min at roomtemp, tipmix 10 times again and go to next step OR place in a 4°C fridge overnight, tipmix 10 times next day
100 µL distilled water 00:02:00 room temperature Overnight at 4°C
2m
Place on a magnetic tube stand until no beads are left in suspension
Take the supernatant with your DNA and put it in a fresh tube