DNA clean up protocol

To remove residual contamination from DNA preparations, or to clean up DNA after PCR and other enzymatic treatments, DNA was cleaned using a spin column protocol from either the Qiaquick PCR purification kit or the Bioline PCR and gel kit. The protocols were carried out according to the manufacturer’s instructions, but in both cases elution of the DNA from the filter column was carried out with water instead of the elution buffer provided with the kits to avoid interference with downstream applications. Additionally, elution steps were performed twice, to increase the yield, with two elutions of 30 µl for the Qiagen kit and 10 µl for the Bioline kit.

2.5 Quantitative PCR.

2.5.1 Reverse Transcription of RNA samples.

In order to generate single-stranded cDNA for qPCR experiments on environmental RNA samples, reverse transcription was carried out using Bioscript reverse transcriptase (Bioline). Reactions were set up with 1 µg RNA template, 0.2 µg of random hexamer primers and DEPC-treated H20 added to a final volume of 12

µl. This mix was incubated for five minutes at 70oC and then chilled on ice. 1 µl 10mM dNTP mix, 4 µl 5x Bioscript reaction buffer, 10 units of RNasin Plus Ribonuclease inhibitor (Promega) and DEPC-treated H20 to 19.5 µl were then added,

vortexed, and finally 0.5µl BioScript was added. The mixture was incubated at 42oC for 60 min, after which it was heated to 70oC for 10 min to stop the reaction and then chilled on ice and stored until use at -20oC. DNA produced in this way was suitable for use in amplification reactions without additional purification.

2.5.2 Assay conditions.

qPCR was performed in an Applied Biosystems StepOnePlus Real-Time PCR system in a 96 well plate format. The reagents used were either Qiagen Quantifast SYBR green PCR kit, or Thermo Scientific SYBR Green qPCR master

mix. Experiments that were to be directly compared were always performed with the same reagents. For both reagents, reactions were performed in 25 µl volumes, and made up with identical quantities of components. Master mixes were made up with components calculated per 25 µl reaction, utilising 2.5 µl each primer (for 0.25 µM), 6.5 µl of SYBR green master mix, 12.5 µl nuclease-free water for each 25 µl final volume. 24 µl of the final master mix and 1 µl of template DNA, either 50 ng of unknown DNA or DNA of known copy number per µl for standard curve production, were added to each well.

2.5.3 Production of standard curves for relative quantification.

Relative abundance was used to enumerate target organisms as a proportion of the total bacterial population present in a sample using universal and group specific primers, or to compare the same target in two differently treated samples. Standard curves were generated as described in Smith et al. (2006) for this purpose. Triplicate serial dilutions ranging from 3 x 108 – 3 x 103 gene copy numbers per µl were prepared for each target gene and for each primer set.

Gene copy numbers were calculated according to the following formula: 100´[concentration of sample in g/µl]

[length of template] ´ 660 ´ 6.022´10 23

where length of template represents either the length of the linearised plasmid or of the PCR fragment.

Triplicate repeats allowed occasional spurious reactions to be disregarded from the analysis, and mean Ct values for each standard curve dilution and each unknown sample could be obtained from duplicate or triplicate values. Ct values were plotted against log gene copy number to generate standard curves for each primer set used in the assay. Linear regression of the data generated equations with which Ct values from unknown samples could be converted to values in terms of gene copy number of the target gene. Copy numbers determined with specific primer sets were expressed as a fraction of the copy number reported by the universal primer set run in parallel. Copy numbers determined when comparing different samples with the same set of primers was carried out by expression the lower sample as a fraction of the higher sample.

In order to generate standard curves, template DNA consisting of 16S rRNA subunit gene sequences of the target Clostridium groups was prepared from strains of E. coli harbouring plasmids containing cloned insert 16S rRNA gene copies, provided by Dr James McDonald (University of Bangor). A strain containing

54 5 4

prepared by PCR amplification of DNA extracted from a pure culture of Fibrobacter succinogenes, also provided by Dr James McDonald.

2.5.3.1 Production of DNA template for standard curve generation.

Plasmid template DNA containing the 16S rRNA gene of Clostridium groups was prepared from overnight cultures of the host E. coli strains in LB. 1.5ml of culture supernatant was centrifuged, the supernatant discarded and plasmids extracted using the Qiaprep spin miniprep kit (Qiagen). Plasmids were linearised by restriction enzyme digestion before use in amplification reactions. All plasmid digestions were performed in 50 µl reaction volumes, digested at 37 oC for 1 h and inspected by agarose gel electrophoresis. For the Fibrobacter group, a PCR product of the 16s rRNA gene was subjected to further rounds of amplification to produce plentiful material for use in qPCR reactions. Table 2.1 summarises the genes used as templates for standard curves

Table 2.1 Source of Standards for qPCR experiments.

Template DNA* Source Purification method

C. aldrichii, for group III clostridia

E. coli strain containing plasmid with cloned C. aldrichii 16SrRNA gene

Miniprep, digestion with

NcoI

C. sporosphaeroides for Group IV clostridia

E. coli strain containing plasmid with cloned C. sporosphaeroides

16SrRNA gene

Miniprep, digestion with

NdeI

C. lentocellum for group XIV clostridia

E. coli strain containing plasmid with cloned C. lentocellum 16SrRNA gene

Miniprep, digestion with

NdeI F. succinogenes for Fibrobacter genus PCR amplification of F. succinogenes 16S rRNA gene.

Size selection through electrophoresis and gel extraction.

*Clostridium groups as defined by Collins et al. 1994; Fibrobacter and F. succinogenes as described in Ransom-Jones et al. 2012