2.3.1 Crude DNA preparation using boiled bacterial colonies
A small portion of a single colony was transferred into 50 µl SDW in a 0.2 ml PCR tube. The suspension was heated to 100˚C for 5 minutes and the resulting template used immediately in the standard PCR.
2.3.2 Standard polymerase chain reaction (PCR)
Standard PCR was carried out in 96 well PCR plates or 0.2 ml PCR tubes, depending on the number of samples. Each reaction contained: 1X Green GoTaq Flexi Buffer, 2.5 mM MgCl2, 0.5 U GoTaq DNA polymerase (all Promega), 10 µM each dNTP
(Bioline), 300 nM each primer and 5 µl crude DNA template. SDW was added to a final volume of 25 µl. Green GoTaq Flexi Buffer contains two dyes that migrate during gel electrophoresis, so addition of loading dye for gel electrophoresis was not required. All primer sequences and PCR cycling conditions are described in Table 2.2. PCR cycling was carried out in an Eppendorf MasterCycler Gradient.
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Table 2.2. Oligonucleotide primers used in this study
Name 5̍→3̍ sequence Product
size (bp)
Target gene/ region
Cycling conditions Study Reference
P2F CTCCACTTCTCGGTTGCTTC 206 LES phage 2 95°C, 4 min then 30
cycles: 95°C, 30 s; 58°C, 30 s; 72°C, 30 s; final extension step, 72°C, 7 min Any PCR assay involving detection of free phages or determining prophage complement of bacterial strain This study P2R ACTAGCCCCGTATCCGAGTT
P3F TCAGGAAAACCTTGCCATTC 384 LES phage 3
P3R GTCTTCTGGTGGTCGGTGAT
P4F AGTTACGCCTGCTGGTGAGT 506 LES phage 4
P4R CCTCAGTCGTGCCTTCTTTC
2totF AGTAGCCGACCCAGACCTTT 141 LES phage 2 95°C, 10 min; then
40 cycles: 95°C, 10 s; 60°C, 15 s; 72°C 30s
qPCR to detect relative copies of free LES phages
(James et al., 2012)
2totR ATGGAAGCAACCGAGAAGTG
3tot1F CGCAGGTACCACCAGACTTT 122 LES phage 3
3tot1R CATGTCCAGCAGGTTCAAAA
4tot1F GCTCATGAGTGGCTGACAAC 105 LES phage 4
4tot1R TCTTGGGCAGAGAACCATTC
PS1 ATGAACAACGTTCTGAAATTCTCTGCT 249 P. aeruginosa oprI
lipoprotein gene
94°C, 5 min then 30 cycles: 94°C, 1 min; 60°C, 1 min; 72°C, 2 min; final extension step, 72˚C, 5 min.
qPCR of free phages; confirmation of degradation of Pseudomonas DNA
(De Vos et al., 1993)
PS2 CTTGCGGCTGGCTTTTTCCAG
lasRF GTGCCGAATCCATATTTG 854 lasR gene 95°C, 4 min then 30
cycles: 95°C, 30 s; 48°C, 30 s; 72°C, 30 s; final extension step, 72°C, 7 min. Amplification and Sanger sequencing of lasR gene (Fothergill et al., 2007) lasRR CCTTCCCTATATATCTGC
7leftF TTCGAGTTGGATCCGGCC 247 fimU LJ (left
phage-host junction)
95°C, 4 min then 30 cycles: 95°C, 30 s; 55°C, 30 s; 72°C, 30 s; final extension step, 72°C, 7 min. Confirmation of LESφ4 integration sites in PAO1 This study 7leftR GTCGTGCTGTGCTGATCTTT
7fimUrF CGCTCATTCCGTGCCAATTA 358 fimU RJ (right
phage-host junction) 7fimUrR TCAATGCGATGCTGCAGTAC
7leftF 328 fimU intact
insertion site 7fimUrR
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11leftF CTTCTTCAAGGCCAAGGGGT 458 pilV LJ
11leftR CGATGGCGATACGGTGATGA
11rightF GACGAGGTGCTTAGACGGAG 610 pilV RJ
11rightR ATCATGGACAGGCCCGATTG
11leftF 349 pilV intact
integration site 11rightR
Nar1F GTATCCGATTCCATCCTGGGGAA Variable 5̍ end of LES
phage 4
94°C, 3 min then 30 cycles: 94°C, 1 min; 60°C, 2 min; 72°C, 4 min; final extension step, 72°C, 7 min. Inverse PCR to determine LESφ4 integration sites This study NarIR GGATGACCACTTGTACGCCAAGC
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2.3.3 Visualisation of PCR products
PCR products were visualised by agarose gel electrophoresis. Molecular grade agarose (Bioline) was dissolved in 0.5X TBE buffer (Appendix B). A 2% (w/v) agarose gel was used for PCR products below 650 bp, and a 1% (w/v) gel used for larger DNA fragments, or visualisation of intact genomic DNA. The agarose was melted by heating and cooled (to avoid vapour formation) prior to the addition of ethidium bromide to a final concentration of 0.1 µg ml-1. 4 µl of PCR product was
added to each well, and a 1 KB+ marker (Invitrogen) was run alongside for size determination of products. For PCR reactions using the colourless GoTaq Flexi Buffer, 5 parts DNA were mixed with 1 part loading dye (Invitrogen), prior to loading. Electrophoresis was performed for 45-90 minutes at 100V and DNA was visualised under an UV transilluminator (Syngene InGenius).
2.3.4 DNA extraction and purification from gels
After agarose gel electrophoresis, required bands were cut from the gel using a disposable scalpel. DNA was extracted from the gel slice using the Qiaquick gel extraction kit (Qiagen). The gel slice was weighed and dissolved in buffer QG (3 volumes buffer to 1 volume gel) by heating to 50˚C for 10 minutes. Isopropanol was added (1 volume isopropanol to 1 volume gel) and the mixture added to a spin column to bind the DNA. The column was centrifuged at 13 000 r.p.m. for 2 minutes and 500 µl buffer QG added to remove traces of agarose. The column was
centrifuged again and 750 µl buffer PE (contains ethanol) was added to wash the DNA. The column was allowed to stand for 2 minutes before centrifugation at 13,000 r.p.m. for 1 minute. The column was centrifuged for a further minute to remove traces of buffer PE and DNA was eluted by pipetting 30 µl buffer EB directly onto the centre of the membrane and allowing to stand for 2 minutes, followed by centrifugation at 13 000 r.p.m. for 1 minute.
2.3.5 Determination of DNA concentration and quality
DNA quality was assessed by spectrophotometric analysis, using a Nanodrop ND- 1000 spectrophotometer. The A230, A260 and A280 of 2 µl of DNA were measured, and
the A260/280 and A260/230 ratios calculated. DNA was deemed acceptable quality if the
45 was estimated by fluorimetric analysis, as this measures only DNA, and is not
influenced by other nucleic acids that may be present in the sample. The double- stranded DNA broad range assay (Invitrogen) was used to estimate DNA
concentration. 10 µl DNA was added to 190 µl Qubit buffer (prepared according to the manufacturer’s instructions), which contains a dye that binds to DNA and emits a fluorescent signal. Samples were vortexed for 2-3 seconds and incubated at room temperature for 2 minutes. The fluorimetric signal was measured using the Qubit fluorometer and DNA concentration was estimated by comparison to known standards.