Apr 12, 2018
Genomic DNA extraction protocol using DNeasy Blood & Tissue Kit (QIAGEN) optimized for Gram-Negative bacteria
- Pavlova A.S.1,
- Olneva T.A.1,
- Konovalova T.A.1,
- Guseva A.N.1,
- Goptar I.A.1,
- Podkolzin A.T.1,
- Kuleshov K.V.1
- 1Central Research Institute of Epidemiology
Protocol Citation: Pavlova A.S., Olneva T.A., Konovalova T.A., Guseva A.N., Goptar I.A., Podkolzin A.T., Kuleshov K.V. 2018. Genomic DNA extraction protocol using DNeasy Blood & Tissue Kit (QIAGEN) optimized for Gram-Negative bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.paadiae
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
We use this protocol and it's working
Created: April 06, 2018
Last Modified: April 12, 2018
Protocol Integer ID: 11298
Keywords: Gram-Negative bacteria, QIAGEN, DNA extraction, DNA elution, proteinase, genomic dna extraction from shigella spp, genomic dna extraction protocol, negative bacteria, dna extraction protocol, genomic dna extraction, shigella spp, salmonella spp, dna extraction, bacteria, genome sequencing, using dneasy blood, genome, dna, dneasy blood
Abstract
We found many specific steps and conditions for gram-negative bacteria while working with DNeasy Blood & Tissue Kit. These details are described in DNeasy Blood & Tissue Handbook (https://www.qiagen.com/us/resources/resourcedetail?id=6b09dfb8-6319-464d-996c-79e8c7045a50&lang=en). However we decided to describe this protocol step by step pointing out in some critical steps. This protocol was successfully applied while genomic DNA extraction from Shigella spp. and Salmonella spp. strains for whole-genome sequencing.
Materials
MATERIALS
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Ethanol
Proteinase KAmrescoCatalog #0706-1G
STEP MATERIALS
Buffer ATL QiagenCatalog #19076
Proteinase KAmrescoCatalog #0706-1G
Buffer AL (lysis buffer)Qiagen
ethanol
Buffer AW1QiagenCatalog #19081
Buffer AW2QiagenCatalog #19072
Buffer AEQiagenCatalog #19077
Buffer AEQiagenCatalog #19077
Buffer ATL QiagenCatalog #19076
Proteinase KAmrescoCatalog #0706-1G
Buffer AL (lysis buffer)Qiagen
ethanol
Buffer AW1QiagenCatalog #19081
Buffer AW2QiagenCatalog #19072
Buffer AEQiagenCatalog #19077
Buffer AEQiagenCatalog #19077
Protocol materials
Buffer AL (lysis buffer)Qiagen
Buffer AEQiagenCatalog #19077
Buffer ATL QiagenCatalog #19076
Buffer AW2QiagenCatalog #19072
Ethanol
Proteinase KAmrescoCatalog #0706-1G
Buffer AW1QiagenCatalog #19081
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Proteinase KAmrescoCatalog #0706-1G
ethanol
Troubleshooting
Safety warnings
Always use Biosafety Level 2 practices (at a minimum) and extreme caution when transferring and handling strains of these genera. Work in a biological safety cabinet when handling large amounts of cells. Disinfect or dispose of all plasticware and glassware that come in contact with the cultures in a safe manner.
It is recommended to plate cultures, prepare cell suspensions, and cast plugs in a Class II Biosafety Cabinet (BSC), if available. Treat all plasticware, glassware, pipets, spatulas, etc. that come in contact with the cell suspensions or plugs as contaminated materials and dispose of or disinfect according to your institutional guidelines.
Before start
Add Ethanol 96% to reagents of QIAGEN kit as described by vendor
Prepare CSB solution (Cell suspension buffer)
1 M Tris-HCl (pH 8.0) - 10 ml
0.5 M EDTA (pH 8.0) - 20 ml
Add deionized water to 1 Litre
Store at +4°C to +8°C
Prepare Proteinase K solution (20 mg/ml) in deionized water
Store at -20°C
Solutions to prepare in advance
Add Ethanol to reagents of QIAGEN kit as described by vendor
CSB solution (Cell suspension buffer)
1 M Tris-HCl (pH 8.0) - 10 ml
0.5 M EDTA (pH 8.0) - 20 ml
Add deionized water to 1 Litre
Store at +4°C to +8°C
Prepare Proteinase K solution (20 mg/ml) in deionized water
Store at -20°C
Ethanol 96%
Plating for confluent growth
Streak an isolated colony from test cultures onto Trypticase Soy Agar (TSA) plates for confluent growth.
Incubate aerobically at 37°C for 18-24 hours.
37 °C incubation
24:00:00 incubation
Preparation of bacterial culture
24-hour culture is suspended in 2.2 ml CSB Buffer and measured the optical density by taking a value of D=7.0 (21х108 cells/ml).
Note
Use a sterile polyester-fiber or cotton swab that has been moistened with sterile CSB to remove some of the growth from agar plate; suspend cells in CSB by spinning swab gently so cells will be evenly dispersed and formation of aerosols is minimized.
We used a densitometer (Densi-La-Meter ® II, Erba Lachema, Czech Republic) for the measurement of bacterial suspension optical density, subsequently brought to a value of D=7.0, which, according to McFarland standards, corresponds to 21х108 cells/ml.
2.2 µL CSB Buffer
Concentration of bacterial cells
Transfer 1 ml of bacterial cell suspension into a microcentrifuge tube and centrifuging for 10 min at 7 500 rpm. Carfully discard supernatant.
1 µL bacterial suspension
00:10:00 centrifugation
DNA Lysis Buffer
Add 180 µl Buffer ATL (QIAGEN) to the pellet and carefully resuspense it.
180 µL Buffer ATL
Buffer ATL QiagenCatalog #19076
Note
You should use 1000 µl pipette tips to prevent cell injury.
Lysis Incubation
Add 20 µl Proteinase K (QIAGEN) or 2 µl of self-prepared Proteinase K (20 mg/ml) (see 'Reagents section'). Mix thoroughly by vortexing, and incubate 1 hours at 56°C.
2 µL Proteinase K
Proteinase KAmrescoCatalog #0706-1G
01:00:00 incubation
56 °C incubation
Note
It is crucial that Proteinase K should be freshly prepared or stored at -200C before use.
DNA precipitation
Vortex for 15 s. Add 200 µl Buffer AL (QIAGEN) to the sample, and pipetting thoroughly up and down to yield homogeneous solution.
200 µL Buffer AL
Buffer AL (lysis buffer)Qiagen
Note
A white gelatinous lysate may form on addition of Buffer AL and ethanol. This lysate can clog membrane pores when the mixture will be placed into the column. To avoid this effect pipette sample thoroughly up and down to yield homogeneous solution.
Then add 200 µl ethanol 96%, and again thoroughly pipetting. Mix the sample by vortexing.
200 µL Ethanol 96%
ethanol
DNA extraction
Pipet the mixture (including any precipitate) into the DNeasy Mini spin column (QIAGEN) placed in a 2 ml collection tube. Centrifuge at 8 000 rpm for 1 min.
00:01:00 centrifugation
Wash
Discard flow-through and collection tube. Place the DNeasy Mini spin column in a new collection tube.
Add 500 µl Buffer AW1 to the column and centrifuge at 8 000 rpm for 1 min.
500 µL Buffer AW1
Buffer AW1QiagenCatalog #19081
00:01:00 centrifugation
Again discard flow-through and collection tube and place the DNeasy Mini spin column in a new collection tube.
Add 500 µl Buffer AW2 to the column. Centrifuge at 14 000 rpm for 3 min.
500 µL Buffer AW2
Buffer AW2QiagenCatalog #19072
00:03:00 centrifugation
DNA elution
Place the DNeasy Mini spin column in a clean 1.5 ml microcentrifuge tube and pipet 100 µl Buffer AE directly onto the DNeasy membrane.
Incubate the sample at room temperature (23-25°С) for 2-5 min, centrifuge at 8 000 rpm for 1 min to elute the DNA solution.
The first elution should contain aproximately 80-90 µl DNA.
100 µL Buffer AE
Buffer AEQiagenCatalog #19077
00:05:00 incubation
25 °C incubation
00:01:00 centrifugation
Place the DNeasy Mini spin column in another clean 1.5 ml microcentrifuge tube and again pipet 100 µl Buffer AE directly onto the DNeasy membrane.
Incubate the sample at room temperature (23-25°С) for 2-5 min and then centrifuge at 8 000 rpm for 1 min.
The second elution contains 100-110 µl DNA.
100 µL Buffer AE
Buffer AEQiagenCatalog #19077
00:05:00 incubation
25 °C incubation
00:01:00 centrfugation