Lambda Phage Manipulations:

2.2.3.1 Preparation of Bacterial Cultures for XFixII Phage

XFixII phage is a replacement vector that can accommodate insert sizes o f DNA ranging from 9-23 kb. The strains SRB and SRB(P2) are the bacterial hosts which have been modified to enhance the stability of clones containing methylated DNA. They also enhance the stability of non-standard DNA structures.

10 ml of L-broth containing 0.2%v/v maltose and 10 mM MgSO^ were inoculated with SRB strain and grown overnight with shaking at 32®C. The SRB E.coli strain restricts the growth o f wild type lambda phage which are spi + phage. Therefore only phage where the recombination genes that give the spi^ phenotype are deleted will be propagated. Maltose induces the expression of maltose binding protein to which the phage will adhere during infection. Phage will also adhere to dead cells so a low temperature will prevent the culture from overgrowing. The cells were pelleted by centrifugation at 2000 rpm at 4®C for 10 minutes. The supernatant was discarded and the pellet was gently resuspended in 10 ml o f 10 mM MgS0 4, then respun at 2000 rpm at 4°C for a further 10 minutes. The supernatant was again discarded and the pellet was resuspended in 4 ml of 10 mM MgSO^. The bacteria was aerated at 32^C

for 45 minutes before being stored at 4®C. The bacteria are stable in this condition for approximately one month.

2.2.3.2 Determination of pfu and Plating of ^.FixII Phage

Serial dilutions of 1 buffer containing XFixII phage were prepared in order to estimate the number o f plaque forming units (pfu) present per ml o f buffer. NZY-Top agar was autoclaved and left in a SO^C to cool. 1 drop of starved bacteria was added to 100 pi of each phage dilution in a tube and inverted 3 times. The tube was incubated at 37®C

for 15 minutes. 4 ml of NZY-top agar was added to the tube and the culture was plated out on NZY plates. Once the agar had set, the plates were incubated at 37^0 for 8 hours which was the optimal time for plaque formation. The number of plaque forming units could then be calculated.

2.2.3.3 High Titre Stock Preparation of XFix II Recombinant Phage

7.5ml o f X buffer was added to a plate containing plaques, preferably in the order of 300-1000 plaques. The plate was gently rocked for at least 1 hour at room temperature. The buffer was then collected in a tube and 1 drop o f chloroform was added. After inverting 2-3 times, the tube was stored at 4®C. The phage would now be viable for at least a year.

2.2.3.4 Isolation of XFix II DNA (Low Multiplicity of Infection)

Two methods could be used for the isolation o f phage DNA. The first method was based on infecting the bacterial culture at a high multiplicity o f infection which would result in complete lysis in 3-5 hours. This method was used if a X phage strain grew well. The second method, which was applicable to the XFix II vector, was the low multiplicity o f infection method which was employed when a slow growing X phage was used, described by Sambrook et al., (1989). This method was modified to optimise the yield obtained for the XFix II vector. The bacterial culture was prepared as described previously and 100 pi was used to inoculate 500 ml o f NZYCM medium in a 5 1 flask. The culture was left to incubate overnight at 25°C (without shaking). A further 500 ml of NZYCM medium was added and IxIO* p.f.u o f high titre phage stock was used to infect the culture. The culture was shaken vigorously at 37®C for 12 hours (or overnight). 20 ml o f chloroform were added and the flask and allowed to shake for a further 10 minutes. A splintery appearance indicated complete lysis. DNAse I and RNAse I were added to a final concentration o f 1 pg/ml and incubated

for 30 minutes at room temperature. NaCl was added to give a 1 M solution (58.4 g/1). The NaCl was dissolved by swirling and left to stand on ice for 1 hour. The addition of salt promotes the dissociation o f the bacteriophage particles from the bacterial debris and was required for the efficient precipitation of the bacteriophage particles from polyethylene glycol. The culture was pelleted by centrifuging at 5000 rpm for 10 minutes at 4®C. The supernatant was transferred to a clean flask where PEG 8000 was added to a final concentration of 10% (w/v), (100 g/1). The PEG 8000 was allowed to dissolve and left to stand on ice for at least 1 hour. The precipitated bacteriophage particles were recovered by centrifugation at 5000 rpm for 10 minutes at 4®C. The pellet was then resuspended in buffer L3 o f the Qiagen Lambda Kit and purified according to the manufacturer's instructions.

2.2.4 Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) is a method of amplifying small amounts of DNA using specific oligonucleotide primers which are extended using the polymerase enzyme from the bacterium Thermus aquaticus, known as Taq polymerase. Reactions were carried out in a 50 pi volume with 200 pM of dATP, dCTP, dTTP and dGTP, 1 pM o f both primers and 1 unit of Amplitaq polymerase and buffer (lOX 100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.1% gelatin (w/v), supplied by Perkin-Elmer Cetus). The PCR was carried out carried out in a Hyhaid Thermal Cycler. Standard thermal cycling conditions included an initial denaturing step, (usually at 94°C), followed by the annealing temperature of the primers (which varied depending on the nucleotide content and the length of the oligonucleotide primer) and an extension temperature of 72°C (the length o f time at this temperature was based on an estimate o f 1 minute per kilobase o f DNA). A total of 30 cycles for each PCR reaction were used. The first cycle: 94°C for 3 minutes 50 seconds; annealing temperature for 2 minutes; 72°C for 2 minutes. Subsequent 30 cycles: 94®C for 1 minute 30 seconds; annealing temperature for 1 minute 30 seconds; 72®C for 1 minute

45 seconds. The final cycle: 94®C for 1 minute 30 seconds; annealing temperature for 1 minute 30 seconds; 72^C for 15 minutes. The final cycling step was used to add dATP to the end of the molecule in order to facilitate T-vector cloning.

2.2.4.1 Vectorette PCR (Riley et aL, 1990)

The development of vectorette PCR has enabled the recovery o f short fragments of DNA adjacent to a known sequence by PCR, for example the ends o f a large genomic clone. The use of known vector sequence to obtain the unknown insert sequence next to it for chromosome walking (Anand et al., 1991), or confirmation o f overlaps in contigs (Coffey et al., 1992) is only one application of this method. With conventional PCR, two different primers at either end of the desired amplified product are required. The advantage o f vectorette PCR is that only one sequence o f the primer pairs is known, usually from the end of the genomic clone. The other end primer will be derived from a vectorette cassette or ‘bubble’. The principle relies initially on obtaining DNA from a clone which can be cosmid, PAC or YAC in origin. The DNA is digested using four restriction enzymes separately, thereby producing four different libraries. The vectorette cassette is then ligated to the ends of each digested library (Figure 2. IB). Hence all fragments will have a vectorette cassette attached to each end. The restriction enzymes used to cleave the DNA can yield either blunt or overhanging ends. The vectorette cassette can also be either blunt-ended or with cohesive overhangs.

The cassette consists of two oligonucleotides, the top strand and the bottom strand (Figure 2.1 A). The end of each strand is complementary, anchoring the two oligonucleotide ends together. In the centre o f both strands is a non-complementary sequence where approximately 29 base pairs do not anneal together. The vectorette primer is designed to the bottom strand of the mismatched region. In this way, a primer from the end of the genomic clone and the vectorette primer can be used in the primary PCR reaction. Thus, in the first cycle, only one strand will be synthesised

Figure 2.1 Vectorette Cassette and Library Construction

A Vectorette cassette. Two complementary oligonucleotides are annealed to each other. A mismatch region in the middle of the two strands will be used to generate a specific primer during the PCR reaction.

B Vectorette library construction. The genomic clone is digested with four restriction endonucleases to generate four different libraries. The vectorette cassette is ligated to the ends o f each library. These libraries will then be used as a template during the PCR reaction.