DNA extraction of bacterial cells using a semi-automated filtration system
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...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2022-01-01
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| Series: | MethodsX |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2215016122001650 |
| _version_ | 1811187534631272448 |
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| author | K.B. Hoorzook T.G. Barnard |
| author_facet | K.B. Hoorzook T.G. Barnard |
| author_sort | K.B. Hoorzook |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T14:04:55Z |
| format | Article |
| id | doaj.art-812ff01bdee04573811fb64f867e448a |
| institution | Directory Open Access Journal |
| issn | 2215-0161 |
| language | English |
| last_indexed | 2024-04-11T14:04:55Z |
| publishDate | 2022-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | MethodsX |
| spelling | doaj.art-812ff01bdee04573811fb64f867e448a2022-12-22T04:19:56ZengElsevierMethodsX2215-01612022-01-019101785DNA extraction of bacterial cells using a semi-automated filtration systemK.B. Hoorzook0T.G. Barnard1Corresponding author.; University of Johannesburg, South AfricaUniversity of Johannesburg, South AfricaThe 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.http://www.sciencedirect.com/science/article/pii/S2215016122001650CeliteEnvironmental waterEscherichia coliGenomic and plasmid extractionGuanidium thiocyanateMembrane filtration |
| spellingShingle | K.B. Hoorzook T.G. Barnard DNA extraction of bacterial cells using a semi-automated filtration system MethodsX Celite Environmental water Escherichia coli Genomic and plasmid extraction Guanidium thiocyanate Membrane filtration |
| title | DNA extraction of bacterial cells using a semi-automated filtration system |
| title_full | DNA extraction of bacterial cells using a semi-automated filtration system |
| title_fullStr | DNA extraction of bacterial cells using a semi-automated filtration system |
| title_full_unstemmed | DNA extraction of bacterial cells using a semi-automated filtration system |
| title_short | DNA extraction of bacterial cells using a semi-automated filtration system |
| title_sort | dna extraction of bacterial cells using a semi automated filtration system |
| topic | Celite Environmental water Escherichia coli Genomic and plasmid extraction Guanidium thiocyanate Membrane filtration |
| url | http://www.sciencedirect.com/science/article/pii/S2215016122001650 |
| work_keys_str_mv | AT kbhoorzook dnaextractionofbacterialcellsusingasemiautomatedfiltrationsystem AT tgbarnard dnaextractionofbacterialcellsusingasemiautomatedfiltrationsystem |