Four methods were used for bacterial DNA extraction from the water, sediment, faeces and pure cultures.
2.5.1.1 DNA extraction from water samples
All buffer solution compositions used in the following methods are listed in Appendix 2. DNA was extracted from the water samples by using a QIAamp DNA mini kit (Qiagen, UK) according to the manufacturer’s protocol (Figure 2.2 a). This method contained four stages; cell lysis, inhibitor removal, protein removal and DNA clean-up (all centrifugation steps were carried out at 13000 x g, unless otherwise stated).
i. Cell lysis
Water samples (100 or 300 ml) were filtered through 0.45 µm pore size filter membranes (Whatman, UK). The filter membrane was transferred in 15 ml tube contained 0.5 ml guanidine thiocyanate buffer (Schulz and Childers, 2011). The filter membrane was mixed well with the buffer and stored overnight at - 20 ºC or for longer at - 80 ºC (Ahmed et al., 2008c). The filtered sample (0.5-1 ml) was placed into a 2 ml Eppendorf tube and then ASL buffer (700 µl) added. The mixture was vortexed for one minute, and then heated at 90 ºC for five minutes, followed by mixing and centrifugation for one minute.
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The supernatant (800 µl) was pipetted into a new 1.5 ml Eppendorf tube; half an InhibitEX® tablet was added to remove inhibitory substances and the mixture vortexed immediately for one minute. The tube was then incubated at room temperature for one minute, followed by centrifugation for three minutes. The supernatant was aspirated and placed into a new 1.5 ml Eppendorf tube, then centrifuged for three minutes.
iii. Protein removal
Proteinase K (15 µl) was added into a new 1.5 ml Eppendorf tube along with 200 µl of the centrifuged supernatant. AL buffer (200 µl) was added. After mixing, incubation was carried out at 70 ºC for 10 minutes, before addition of 200 µl of molecular biology grade ethanol (Fisher BioReagents®, UK).
iv. DNA clean-up
The entire supernatant was pipetted into a QIAamp spin column/ collection tube and centrifuged for one minute. A QIAamp column was placed into a new collection tube and AW1 buffer (500 µl) added, and then centrifuged for one minute. A QIAamp column was placed into a new tube and AW2 buffer (500 µl) added, and then centrifuged for three minutes. A QIAamp column was placed into a new tube (1.5 ml) and elution buffer (AE, 100 µl) added, and then centrifuged for one minute. DNA pellet was transferred into a new 1.5 ml Eppendorf tube and stored at - 20 ºC for future use.
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2.5.1.2 DNA extraction from sediment samples
A SoilMaster™ DNA extraction kit (Cambio, UK) was used for DNA extraction from the sediment samples as described the manufacturer’s protocol (Figure 2.2 b). In brief:
i. Sediment samples (200 mg) were weighted and placed into a 2 ml sterile Eppendorf tube. A soil DNA extraction buffer (250 µl) was added, then followed by proteinase K (2 µl).
ii. Soil lysis buffer (50 µl) was added, the mixture vortexed briefly, and incubated at 65 ºC for 10 minutes. The mixture was then centrifuged at 1000 x g for two minutes.
iii. Supernatant (180 µl) was transferred into a new 1.5 ml Eppendorf tube; protein precipitation reagent (60 µl) was added and mixed thoroughly by inverting the tube 6-8 times. After that, the mixture was incubated on ice for 8 minutes, followed by 8 minutes centrifugation. iv. Following centrifugation, 100 µl supernatant was transferred carefully
into the spin column, followed by further centrifugation for two minutes at 2000 x g in a 1.5 ml Eppendorf tube. The spin column tube was discarded.
v. DNA precipitation solution (6 µl) was added and the mixture incubated at room temperature for 5 minutes. Thereafter, the mixture was centrifuged for 5 minutes and the supernatant decanted carefully. The DNA pellet was washed by addition of 500 µl of pellet wash solution. Finally, 100 µl of TE buffer was added to re-dissolve the pelletted DNA.
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Figure 2.2 The main steps of DNA extraction, (a) water and faecal samples
using QIAamp DNA mini kit and (b) sediment samples using SoilMaster™ DNA extraction kit (adapted from literature supplied by Qiagen and SoilMaster™, UK).
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2.5.1.3 DNA extraction from faecal samples
Faecal samples (200 mg) from human, cow, sheep, horse, dog, deer, pig, cat and duck were weighed and DNA extracted by using the QIAamp stool DNA kit (Qiagen, UK) as described previously for water samples, with some modifications. Briefly, 500 μl lysozyme solution (Sigma Aldrich, UK; 50 mg lysozyme was dissolved in one ml TE buffer) was added to each faecal sample tube, and the mixture incubated at 37 ºC for 30 minutes. In the final step, 100 µl of the AE buffer was added to suspend the DNA pellet and stored at - 20 ºC for future use.
2.5.1.4 DNA extraction from pure culture of Bacteroides
Pure cultures of isolated Bacteroides species were grown in BPRM broth (15-25 ml) as described in section 2.4.1.3. DNA was extracted as described below:
i. One millilitre of broth culture was placed in a 1.5 ml Eppendorf tube, centrifuged at 12000 x g for 3 minutes, and the supernatant discarded. ii. The pellet was washed twice by the sterile PBS (500 µl), and then centrifuged at 12000 x g for 3 minutes. The supernatant was discarded.
iii. TLE lysis buffer (500 µl) was added, and mixed gently.
iv. The mixture was boiled for 10 minutes, and then centrifuged at 12000 x g for 5 minutes.
v. The supernatant was pipetted off carefully and placed in a new 1.5 ml Eppendorf tube.
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vi. 500 µl of Phenol-Chloroform-Isoamyl alcohol (25:24:1 v/ v, kept at 4 ºC) was added. The mixture was mixed, and then centrifuged at 12000 x g for 5 minutes.
vii. The supernatant containing the bacterial DNA was taken and placed in a new 1.5 ml Eppendorf tube, before storage at - 20 ºC.
Extracted DNA from each sample was processed in aliquots to reduce the effect of repeated freeze-thawing. DNA aliquots were kept in ice between reaction steps.