Four molar sodium acetate

8.2g o f sodium acetate were dissolved in 20mL o f water and 1 mL o f glacial acetic acid added. The solution was made to 25mL with H2O.

n-5.2.3.

NaCl 5.8g.L''

MgSOj.VHzO 2.g.L'‘

IM Tris.HCl 5mL.L‘'

2%(w/v) Gelatine 5mL.L''

II-5.2.4. TE-RNase solution

Tris.HCl pH 7.5 lOmM

NaiEDTA ImM

RNasel 20pg.mL'^

II-5.2.5. SSC (standard saline citrate) (x20)

NaCl 3M

Sodium citrate 0.3M

II-5.2.6. THE buffer

Trizma base 90mM

Na.Borate 90mM

Na^EDTA ImM

Ethidium bromide 0.05p.g.mL'^

II-5.2.7. DNA sample loading buffer

EDTA O.IM

Sucrose 40% (w/v)

Bromophenol blue 0.15m g.m L'‘

II-5.2.8. Denhardt’s Solution (x50)

Ficoll lOg.L-'

B.S.A. 10g.L‘‘

M aterials and M eth o d s Chapter II

II-5.2.9. Pre-hybridisation buffer

s e e x6

Denhardt’s solution x5

SDS 0.5%(w/v)

Denatured Sperm DNA lOOpg.mL*'

II-5.3. Agarose gel electrophoresis

Gel electrophoresis was performed using gel concentrations from 0.7 to l%(w/v) agarose. For a l% (w /v) agarose gel, Ig o f agarose was added to 100 mL o f TBE buffer and dissolved by boiling. Molten agarose was poured onto glass plates surrounded by masking tape and a comb inserted 10mm from the top o f the plate. Once set, the autoclave tape and comb were removed and the gel submerged in Ix TBE buffer. The gels were electrophoresed at 200V for 1-2 hours or overnight at 20V.

II-5.4. Restriction digestions and ligations

Restriction digestions were performed by the method o f Sambrook et a i, 1989. All reactions were carried out at 37°C in the appropriate buffer (Pharmacia “one-Phor- all”) concentration. Ligations were performed overnight at 4°C unless otherwise stated. Aliquots were tested for digestion or ligation by eleclrophoresing control samples in parallel on agarose gels.

II-5.5. DNA preparation

11-5.5.1. Large scale preparation of

Bacillus

1 g o f cell paste was resuspended in 2mL TE buffer, a few crystals o f lysozyme were added, and the solution incubated at 37°C for 15-30 minutes. Following incubation, O.lmL o f 10% SDS and O.SmL o f Pronase (lOmg.mL'*) was added, gently mixed and incubated for 1 hour at 37°C. To this, 5mL o f TE-RNase buffer was added and incubated at 3 T C for 1 hour. 8 mL o f buffered phenol was then added, gently mixed for 5 minutes and centrifuged for 15 minutes at 1000 x g. The top layer was removed to a clean container and 6mL of phenol (25): chloroform (24): isoamyl alcohol (1) mixture added and mixed. The mixture was centrifuged at 4000x g for 15 minutes and the top phase again removed.

M aterials and M eth od s Chapter II

To this, an equal volume o f chloroform (24); isoamyl alcohol (1) was added, mixed for 5 minutes and then centrifuged at 4000x g for 15 minutes.

The top layer was again removed and the DNA spooled after the addition o f 0.1 volumes o f 4M sodium acetate and the layered addition o f 2 volumes o f 100% ethanol. The spooled DNA was resuspended in 2mL TE buffer and precipitated overnight with 0.1 volumes of sodium acetate and 2 volumes o f 100% ethanol.

The DNA was pelleted, washed with 70%(v/v) ethanol and resuspended in TE buffer. The 260/280 absorbance ratio was then determined.

II-5.5.2. Small scale preparation of plasmid DNA

Plasmid DNA was isolated from 5mL nutrient broth cultures grown overnight in universal bottles and purified using Qiagen mini prep columns.

II-5.5.3. Small and large scale phage preparation

Phage lysate preparation - 2mL

MRA (P2) cells were grown overnight at 30^C at ISOrpm in 5mL o f LB broth. At the same time single plaque titres were prepared for use (dilutions were prepared on the basis that 1 plaque = 10^ phages). Cells were harvested at 1500xg for 10 minutes and resuspended in 2.5mL o f lOmM MgS0 4. O.SmL aliquots were dispensed into 5

universal tubes and 1-5 well isolated plaques (excised as agar plugs) added from the phage titre plates. The tubes were vortexed and allowed to stand at room temperature for 5 minutes, before addition o f 2mL LB broth containing lOmM M gS0 4 and vigorous

agitating at 37°C for 4-6 hours or until lysis occurred. As lysis occurred, O.lmL o f chloroform was added and the lysate was then centrifuged to remove the cell debris.

Phage lysate preparation -1 litre

50mL o f MRA (P2) was cultured overnight at 30"C in LB containing lOmM MgSÛ4. 20mL o f this overnight culture was then mixed with approximately 5 x 10^

M aterials and M eth o d s C hapter II

was then added to 1 litre o f pre-warmed LB containing lOmM M gS0 4 and incubated at dT C with vigorous agitation. After 5-7 hours incubation, lysis occurred and solid NaCl

was added to a final concentration o f 0.5M (29g.L’’) together with ImL o f chloroform. This was then centrifuged at 600x g for 10 minutes to pellet cell debris.

II-5.5.4. Large scale isolation of phage DNA

lOg o f PEG 6000 were added to every lOOmL o f lysate and stirred at room temperature until the PEG dissolved completely. This was placed on iced water for at least 60 minutes, causing the co-precipitation o f PEG and DNA. The precipitate was collected by centrifugation at 6000g for 10 minutes at 4°C, resuspended in 20 mL of 50mM Tris.HCl pH 7.5, lOmM MgS0 4 and the PEG-phage suspension extracted with

an equal volume o f chloroform by gently inverting for 1 hour. The extraet was eentrifuged at 2000x g to separate the phases, and the top (aqueous) phase eontaining the phage partieles was removed and subjected to CsCl gradient separation.

II-5.5.5 CsCl gradient centrifugation

After the extraction with chloroform, the aqueous phase was removed and 0.75g o f CsCl was added per mL o f bacteriophage suspension and mixed gently to dissolve. The solution was transferred to an ultraeentrifuge tube filled to the top with SM buffer containing CsCl 0.75g.mL"', heat sealed and centrifuged at 38,000rpm for 24 hours at 4°C (Beckman Ti50 rotor). Following centrifugation the bacteriophage could be seen as a blue-white band, which was recovered using a syringe and needle.

The extracted band was diluted 3x with SM buffer and phenol/chloroform extracted with an equal volume. This was centrifuged at 1500x g for 10 minutes and the top layer removed. To this layer, 0.1 volumes o f 4M Na-acetate and 2 volumes of ethanol was added and ineubated overnight at -20"C. The phage pellet was resuspended in SM buffer.

11-5.5,6. Size fractionation

oiBacillus

Trial digestions o f genomic Bacillus strain RAPcS DNA with SauSAl were carried out to determine the optimum conditions to produce the 9 to 23kb DNA fragments. 150pg o f genomic DNA was diluted with TE and restriction buffer, and 2

M aterials and M ethods Chapter II

units of Sau3AI added to give a final volume o f 0.2mL. This was incubated at 37°C and 20p.L samples were removed at various time intervals and stop mixture added. The samples were electrophoresed overnight at 20V in parallel on a 0.7%(w/v) gel with the appropriate markers. The DNA fragments with the correct size were excised from the gel, electro-eluted and precipitated overnight. The pellet was resuspended in 10-20p.l o f TE buffer.

n.5.6.

5mL nutrient broth containing 20mM M gS04 was inoculated with fresh E.coli

(JM107) cells from a plate, incubated overnight at 3 T C in an orbital shaker and used to inoculate 2Q0mL o f nutrient broth containing 20mM MgS0 4. The cells were grown to

mid exponential phase (approximately 3-4 hours) at 1>TC in an orbital shaker, harvested in sterile centrifuge pots at 4°C at 10,000 rpm (Sorval GSA rotor) and washed in 40mL ice cold 75mM CaCE containing 15% glycerol. The cells were re-harvested by centrifugation at 4°C and resuspended in 5mL o f ice cold 75mM CaCE , 15% glycerol before aliquoting as 0.2mL samples and storage at -70"C.

II-5.7. Transformation and plating of E.

coli