Cosmid Library Preparation .1 Preparation of insert gDNA

High molecular weight P. alba gDNA was partially digested with Sau3AI (Roche). 50 µg gDNA were digested with 0.025 units of Sau3AI for 30 seconds in a volume of 250 µl. The reaction was immediately quenched with EDTA. The digested DNA was extracted by adding 500 µl phenol-chloroform-isoamylalcohol pH 8 and gently mixing on a rotary mixer.

The phenol and aqueous phases were separated by centrifugation at 13000 rpm for 4 min at 4 °C. The aqueous phase was moved to a fresh tube using a wide-bore tip. The DNA extraction repeated with 500 µl chloroform. The tube contents were mixed slowly by a rotary mixer, then spun at 13000 rpm for 4 min at 4 °C. Sodium acetate was added to the supernatant to a final concentration of 0.3 M. The DNA was precipitated with 500 µl ice-cold isopropanol. DNA was pelleted at 13000 rpm for 4 min at 4 °C. The pellet was washed in 70% ethanol by gentle inversion. The DNA was air-dried and resuspended in 25 µl elution buffer heated to 50 °C. The partially digested gDNA was dephosphorylated with calf intestinal alkaline phosphatase (CamBio) in 50 µl total reaction at 37 °C for 1 hour. The reaction was immediately quenched with EDTA then the partially digested dephosphorylated DNA was extracted with phenol-chloroform-isoamylalcohol pH 8 the chloroform and precipitated with isopropanol as described above. The DNA was air-dried and resuspended in 25 µl elution buffer heated to 50 °C. The partially digested and dephosphorylated DNA was checked on PFGE. The partial Sau3AI digestion reduced the gDNA from 100–200 kb to 30-50 kb in size for ligation into the SuperCosI vector.

2.11.2 Preparation of pSuperCos1 vector and ligation with insert DNA

10 µg SuperCosI vector (Stratagene) were linearised with 9 U/µg XbaI (Roche) in a total volume of 100 µl at 37 °C for 1 hour. Complete digestion was indicated by a linear SuperCosI band at 7.9 kb when 1 µl of the digestion was checked on a 1 % TBE agarose gel. The vector DNA was purified from the remaining reaction with the Qiagen PCR purification kit. The DNA was eluted twice in 25 µl elution buffer warmed to 50 °C. The purified XbaI digested DNA was treated with 30 U calf intestinal alkaline phosphatase

96 (Cambio), in 100 µl total reaction for 1 hour at 37 °C. The DNA was purified with the

Qiagen PCR purification kit and eluted twice in 25 µl elution buffer warmed to 50 °C. The DNA was digested with 10 U/µg BamHI (NEB) in 100 µl total volume at 37 °C for 1 hour.

The DNA was purified with the Qiagen PCR purification kit and eluted with twice in 25 µl elution buffer warmed to 50 °C. The generation of fragments of the expected sizes of 1.3 kb and 6.6 kb was checked by running 2 µl of the treated DNA and alongside the 7.9 kb XbaI linearised DNA on a 1 % TBE agarose gel by electrophoresis. Ligation was carried out as follows with the reaction incubated at 4 °C overnight

Sample Negative Control

Insert DNA Sau3AI digested 2.5 µg 2.5 µg

pSuperCosI BamHI digested 1 µg 1 µg

10 x T4 ligase buffer (Promega) 2 µl 2 µl

T4 DNA ligase 3U/µl (Promega) 1 µl -

H2O to 20 µl to 20 µl

2.11.3 Phage packaging

4 µl of each ligation reaction were packaged into λ-phage using the GigaPack III Gold Packaging Extract (Stratagene) according to the manufacturer’s instructions. Briefly, packaging extracts were removed from -80 °C storage to dry ice. Extracts were rapidly thawed and 4 µl of each ligation reaction immediately added. The extract was gently mixed and incubated at room temperature for 2 hours. 500 µl SM buffer were added, then 20 µl chloroform were also added. Packaged phage were stored at 4 °C and used within 1 month.

2.11.4 Phage titration

Escherichia coli XL-I Blue MR (Stratagene) was streaked from a glycerol stock at -20 °C on to LB agar and was grown at 37 °C overnight. A single colony from this plate was used to inoculate 10 ml LB broth and grown overnight at 37 °C. 1 ml of this culture was used to inoculate 50 ml LB broth containing 10 mM MgSO₄ and 0.2 % maltose. This culture was grown at 37 °C for 3 hours. Cells were recovered by centrifugation and diluted to an OD600

of 0.5 in 10 mM MgSO4. Packaged phage were diluted in SM buffer to a totally volume of 25 µl. Transfection of E. coli cells with 1 µl of 1/5, 1/10, 1/50 and 1/100 dilutions of phage was carried out by mixing 25 µl cells with 25 µl each dilution and incubating cells at room temperature for 30 min. 200 µl LB broth were added to each tube and incubated at 37 °C

97 for 1 hour with gentle shaking every 15 min. The entire transfection reaction was plated

out on LB agar containing 100 µg/ml carbenicillin and incubated overnight at 37 °C. The resulting colonies were counted to give the phage titre per µl of phage extract used. The resulting clones were picked into 5 ml LB broth and grown at 37 ºC overnight. The packaged extract was estimated to contain 20 colony forming units per µl of undiluted extract.

2.11.5 Phage transfection of E. coli to construct cosmid library

This was carried out as a scaled-up version of the phage titration. It was calculated that in order to obtain the 3072 colonies needed to make the library, 225 µl of the packaged extract were transfected to construct the final cosmid library. 500 µl E. coli XL-I Blue MR cells at OD600 of 0.5 in 10 mM MgSO4 were added to 225 µl of the pooled packaged phage extracts made up to 500 µl with SM buffer. 5 ml LB broth were added and incubated at 37 °C for 1 hour with gentle mixing every 15 min. 500 µl of the transfection mix were plated out on each of 6 Genetix plates (240 x 240 x 20 mm) containing 250 ml LB agar with 100 µg/ml carbenicillin to give an estimated plating density of 750 colonies per plate. Plates were incubated inverted overnight at 37 °C.

2.11.6 Library picking and transfer to membrane

3072 colonies were picked using Q-Bot (Genetix) into 8 x 384 well archive plates containing freezing broth (LB and glycerol) and 100 µg/ml carbenicillin. Two copies of the archive plates were replicated and all copies of the library were stored at -80 °C. The entire library was spotted two-fold onto nylon membrane in a double off-set pattern. The clones on the membrane were grown on LB agar overnight and baked on to the membrane at 80 °C. The membrane was stored at -20 °C.

2.11.7 Probe preparation and library hybridisation

Three probes were amplified from P. alba gDNA by PCR using three pairs of primers.

1289FlanA and 1289RlanA primers amplify 230 bp of pspA. 3088F and 3088R amplify 547 bp of pspB. PMSLanAf and PMSLanBf amplify 1701 bp spanning pspA and 5’ pspB.

The PCR products were separated by electrophoresis on a 1 % TBE agarose gel, purified by PCR purification and ligated into pGEM-T vector (Promega) by TA cloning according to the manufacturer’s instructions. 2 µl of the ligation were used to transform DH5α cells and

98 plated onto LB containing 100 µg/ml carbenicillin, 0.5 mM IPTG and 80 µg/ml X-Gal. One

white colony of each insert cloned in pGEM-T was grown in liquid LB broth containing 100 µg/ml carbenicillin. The vector was mini-prepped from the cell pellet using a Qiagen kit. A restriction digest with EcoRI revealed the correct sized insert was present in each vector.

The insert was amplified out of the vector and purified by gel-extraction with a Qiagen kit and the concentration measured using Nanodrop (Thermo Scientific). Each amplified probe was sequenced and found not to contain any mutations. 25 ng of the purified DNA fragment were diluted to a final volume of 50 µl in TE. The DNA was denatured at 95-100

°C for 5 min and snap cooled on ice for 5 min before centrifuging briefly. The DNA was added to a tube containing Rediprime II random prime labelling reaction mix (Amersham), to which was then added 5 µl ³²P-αdCTP and mixed by pipetting. The tube was incubated at 37 °C for 30 min and the reaction stopped by adding 5 µl 0.2 M EDTA. The tube was heated to 100 °C for 5 min then snap cooled on ice for 5 min before centrifuging briefly.

Three copies of the membrane containing the cosmid library clones were prepared by soaking for 1 hour at 42 °C in a pre-hybridisation solution of: 5 x SSC, 0.5 % SDS, 1 mM EDTA (pH 8). The solution was replaced with fresh pre-hybridisation solution and soaked for a further 2 hours. The bacterial debris were scraped off using a damp paper towel and the membranes were rinsed twice in 2 x SSC.

The membranes were each placed into a large hybridisation tube containing 50 ml of hybridisation buffer warmed to 60 °C and were pre-hybridised for 5 hours at 65 °C in a Techne rotisserie oven. The pre-hybridisation solution was replaced with 10 ml of fresh hybridisation buffer. To each of the three tubes, 30 µl of one labelled probe were added.

The filter was hybridised at 60 °C in a Techne rotisserie oven for a total of 20.5 hours. The hybridisation solution was removed and the filter washed five times in 30 ml of pre-warmed wash buffer at 60 °C until the counts per minute were reduced to ~20. The filter was wrapped in clingfilm and exposed to a phosphor plate overnight. The plate was visualised using a phosphorimager (Fuji). Alignment of the membrane with the grid pattern of the 386 well plates along with the double off-set pattern of the spots allowed the location of the positive clones to be identified. Positive clones were selected from the library and grown on LB agar containing 100 µg/ml carbenicillin.