DNA extraction of bacterial cells using a semi-automated filtration system

• Published in: MethodsX 2022-01, Vol.9, p.101785-101785, Article 101785
• Main Authors: Hoorzook, K.B., Barnard, T.G.
• Format: Article
• Language: English
• Subjects: Celite; Environmental water; Escherichia coli; Genomic and plasmid extraction; Guanidium thiocyanate; Membrane filtration; Method; Semi-automated filtration system
• ISSN: 2215-0161; 2215-0161
• DOI: 10.1016/j.mex.2022.101785

The COVID-19 pandemic lockdown created problems with importing of commercial kits resulting in extended turnaround times for consumable deliveries. One way to circumvent this was to use an inexpensive optimized in-house method for DNA extraction from water. • The DNA extraction methods were optimized on a 96-well plate using a semi-automated filtration system to increase the number of samples from 24 to 96 at a time in 2 h. The DNA extraction method optimizations included: (a) Guanidium thiocyanate method plus dilution series of celite to determine DNA binding capacity; (b) QIamp 96 Qiacube HT kit (Qiagen®); (c) Guanidium thiocyanate with the celite replaced with a binding buffer. • The in-house DNA extraction methods and adapted in-house DNA extraction method were compared to QIamp 96 Qiacube HT kit (Qiagen®), which is used on a 96-well semi-automated filtration system. The results showed maximum capacity of the 96-well filter plates was 400 μℓ broth (OD600 = 0.45 = 3.6 × 108 cells/mℓ) before the 96-well filters blocked. • When the methods were compared, there was no significant difference between the in-house DNA extraction method with 1:420 celite dilution (P-value = 0.126) and the adapted in-house method with binding buffer (P-value = 0.298) DNA yield or amplification of PCR products. [Display omitted]