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2.2.3.5.1 For screening of plasmid DNA from both £. ct?// and P. aeruginosa strains

or isolation of plasmid DNA for transformation into E . collyroutinely a modification of

the alkaline lysis method, as described in Sambrook et a l (1989), was used:

1.5 ml of an overnight culture was centrifuged for 5 min., 14,000 rpm in an Eppendorf centrifuge. The supernatant was discarded and the cell pellet resuspended by vortexing in 0.1 ml of an ice-cold solution of 50mM glucose, lOmM EDTA, 25mM

Tris.HCl pH 8.0 (Solution I). When isolating plasmid DNA from P. aeruginosa

strains 4 mg/ml lysozyme was added to Solution I prior to use. After addition of 0.2 ml of a solution of 0.2N NaOH, 1% SDS (Solution H), the tube contents were mixed by inversion several times. Then 0.2 ml of ice-cold Solution HI (60 ml 5M potassium

acetate, 11.5ml glacial acetic acid, 28.5ml H2O) was added and the tube vortexed in an

inverted position for 10 sec. Following 20 min. incubation on ice the tube was centrifuged at 14,000 rpm for 10 min. in an Eppendorf centrifuge and the clear supernatant transferred to a second tube. Contaminating protein was removed by phenol/chloroform extraction (2.2.3.3) and the plasmid DNA precipitated with either ethanol or isopropanol (2.2.3.1/2.2.3.2). DNA pellets were resuspended in 20-30 |il TE buffer (2.1.4.3) and RNase added to a final concentration of 20 pg/ml.

2.2.3.5.2 To improve the yield of both larger plasmids and low copy number

plasmids, especially from P. aeruginosay either the Promega W izard^M Minipreps DNA

Purification system or the Quaigen Plasmid Mini kit were used as per the manufacturers’ instructions.

2.2.3 6 Large Scale Isolation o f Plasmid DNA and CsCl Density Centrifugation

Large scale isolations of plasmid DNA were carried out using a modification of the alkaline lysis procedure (2.2.3.5.1) and the plasmid DNA purified by CsCl density centrifugation. Routinely, 500 ml of an overnight culture was harvested by

centrifugation in a Sorvall RC5-B centrifuge at 5,000 rpm, 4 0C for 10 min. 20 ml of

Solution I containing 4 mg/ml lysozyme was added, the cells resuspended by vortexing and then incubated at room temperature for 5 min. 50 ml of Solution II was added, the

tube contents mixed by inversion several times and left on ice for 5 min. 50 ml of Solution III was added and the mixture inverted several times to mix. After 5 min. on ice the precipitated chromosomal DNA was pelleted by centrifugation in a Sorvall RC5- B centrifuge at 7,000 rpm, 40C for 30 min. The supernatant was transferred to a fresh tube, 50 ml of 50% polyethylene glycol (PEG) 6000 was added and the mixture was placed on ice for 60 min. The precipitated plasmid DNA was pelleted by centrifugation for 30 min. at 10,000 rpm, 40C and resuspended in 5 ml of TE buffer. The DNA solution was phenol/chloroform extracted twice and the solution made up to 9 ml with TE buffer pH 8.0. 1.1 g CsCl per 1 ml of solution and 0.2 ml of a 10 mg/ml solution of ethidium bromide was added. The mixture was centrifuged in Beckman quick-seal polyallomer tubes at 50,000 rpm for 20 hours in a Beckman L-7 ultracentrifuge at 200C. During the final 30 min., the speed of the rotor was reduced to 40,000 rpm to relax the gradient. The plasmid DNA band was removed by inserting a hypodermic needle through the side of the tube and retrieving the DNA band. 4 ml of TE buffer pH 8.0 was added to the DNA solution (approximately 1 ml) to avoid precipitation of the CsCl. The ethidium bromide was removed by repeated extraction with an equal volume of CsCl saturated isopropanol. Finally, the aqueous phase containing the plasmid DNA was ethanol precipitated and resuspended in 0.5 ml of TE buffer.

2.2.3.T R e stric tio n Enzym e D igests

Restriction enzymes were purchased from (Gibco BRL, Paisley, UK; New England Biolabs, Mass., USA). All restriction enzyme digests were carried out using the manufacturers’ recommended buffer. Typically, 20 units of restriction enzyme were used per digest with an incubation period of between 1-3 hours. Reactions were terminated by the addition of 6x stop solution (2.2.3.10) if the digests were to be analysed by electrophoresis, or heat inactivation at 650C for 15 min. if the DNA was to be used for subsequent digests or cloning.

2.2.3.S DNA M odification R eactions

2.2.3.8.1 C onversion of F rag m e n ts with P ro tru d in g 5 ’ Ends to Blunt Ends

Protruding 5 ’ ends were filled in using the polymerisation activity of the Klenow

fragment of E. coli DNA Polymerase I by a modification of the protocol of Sambrook

et al. (1989). The DNA was incubated at room temperature for 15 min. in a final

volume of 20 \ii containing 2 |il lOx buffer (0.5M Tris.HCl pH 7.2, O.IM MgS0 4,

units) of the Klenow fragment (New England Biolabs, Mass., USA). The reaction

was teiTninated by heat inactivation (75^C, 10 min.). The DNA was extracted with

phenol/chloroform and precipitated with either ethanol or isopropanol before resuspension in TE buffer.

2.2.3.8.2 Conversion o f Fragments with Protruding 3 ’ Ends to Blunt Ends

Protruding 3’ ends were made blunt ended using the 3 ’ exonuclease activity of

bacteriophage T4 DNA Polymerase by a modification of the protocol of Sambrook et

a l (1989). The DNA fragment was incubated for 15 min., 120C in a final volume of

20 p.1 containing 2 |il lOx T4 Polymerase buffer (0.33M Tris.acetate pH 7.9, 0.66M potassium acetate, O.IM magnesium acetate, 5mM DTT, 1 mg/ml BSA), 1 pi of a 2mM solution of all four dNTPs and 2 p i (6 units) of T4 DNA Polymerase (New England Biolabs, Mass., USA). The reaction was terminated by heat inactivation (750C, 10 min.). The DNA was purified by phenol/chloroform extraction, ethanol precipitated and resuspended in TE buffer.

2 2.3.9 Ligation of DNA Fragments

Cohesive and blunt end DNA ligations were carried out under similar conditions. Routinely, ligations were carried out in a final volume of 20 pi containing 2 pi of the manufacturers’ recommended lOx ligation buffer and 1 pi (10 units) T4 DNA Ligase (New England Biolabs, Mass., USA) at either 40C (cohesive ends) or Ib^C (blunt ends) overnight. The ligation mixes were usually frozen at -200C for 30-60 min.

before transforming the appropriate E. coli strain.

2 .2 .3 .1 0 Agarose Gel Electrophoresis of DNA

The percentage agarose used was determined by the size of the DNA fragments to be

separated according to Sambrook et a l (1989). The agarose was dissolved in 150 ml

Ix TBE (2.1.4.2) by boiling in a microwave and ethidium bromide was added to a final concentration of 1 pg/ml. Molton agarose was poured onto glass plates surrounded in autoclave tape and a comb inserted 10 mm from the top of the plate. Once set, the autoclave tape and comb were removed and the gel submerged in 500 ml Ix TBE. DNA samples were mixed with an appropriate volume of 6x stop solution (40% (w/v) sucrose, O.IM EDTA, 0.015 mg/ml bromophenol blue dye) and loaded into the wells.

The gels were electrophoresed at the required voltage during the day or 20-30v overnight.